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Sample records for human aml cells

  1. Telomerase inhibition effectively targets mouse and human AML stem cells and delays relapse following chemotherapy

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    Bruedigam, Claudia; Bagger, Frederik Otzen; Heidel, Florian H.

    2014-01-01

    Acute myeloid leukemia (AML) is an aggressive and lethal blood cancer maintained by rare populations of leukemia stem cells (LSCs). Selective targeting of LSCs is a promising approach for treating AML and preventing relapse following chemotherapy, and developing such therapeutic modalities is a k...

  2. Oncogenic fusion proteins expressed in immature hematopoietic cells fail to recapitulate the transcriptional changes observed in human AML

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    Rapin, N; Porse, B T

    2014-01-01

    in acute promyelocytic leukemia. Hematopoietic stem/progenitor (HSPCs) cells transduced with oncogenic fusion genes are regarded as promising in vitromodels of their corresponding AML subtypes. Here, we critically assessed the potential of such in vitro models using an integrative bioinformatics approach...

  3. Mitochondrial BAX Determines the Predisposition to Apoptosis in Human AML.

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    Reichenbach, Frank; Wiedenmann, Cornelius; Schalk, Enrico; Becker, Diana; Funk, Kathrin; Scholz-Kreisel, Peter; Todt, Franziska; Wolleschak, Denise; Döhner, Konstanze; Marquardt, Jens U; Heidel, Florian; Edlich, Frank

    2017-08-15

    Purpose: Cell-to-cell variability in apoptosis signaling contributes to heterogenic responses to cytotoxic stress in clinically heterogeneous neoplasia, such as acute myeloid leukemia (AML). The BCL-2 proteins BAX and BAK can commit mammalian cells to apoptosis and are inhibited by retrotranslocation from the mitochondria into the cytosol. The subcellular localization of BAX and BAK could determine the cellular predisposition to apoptotic death.Experimental Design: The relative localization of BAX and BAK was determined by fractionation of AML cell lines and patient samples of a test cohort and a validation cohort.Results: This study shows that relative BAX localization determines the predisposition of different AML cell lines to apoptosis. Human AML displays a surprising variety of relative BAX localizations. In a test cohort of 48 patients with AML, mitochondria-shifted BAX correlated with improved patient survival, FLT3-ITD status, and leukocytosis. Analysis of a validation cohort of 80 elderly patients treated with myelosuppressive chemotherapy confirmed that relative BAX localization correlates with probability of disease progression, FLT3-ITD status, and leukocytosis. Relative BAX localization could therefore be helpful to identify elderly or frail patients who may benefit from cytotoxic therapy.Conclusions: In this retrospective analysis of two independent AML cohorts, our data suggest that Bax localization may predict prognosis of patients with AML and cellular predisposition to apoptosis, combining the actual contribution of known and unknown factors to a final "common path." Clin Cancer Res; 23(16); 4805-16. ©2017 AACR. ©2017 American Association for Cancer Research.

  4. Isoform-specific potentiation of stem and progenitor cell engraftment by AML1/RUNX1.

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    Shinobu Tsuzuki

    2007-05-01

    Full Text Available AML1/RUNX1 is the most frequently mutated gene in leukaemia and is central to the normal biology of hematopoietic stem and progenitor cells. However, the role of different AML1 isoforms within these primitive compartments is unclear. Here we investigate whether altering relative expression of AML1 isoforms impacts the balance between cell self-renewal and differentiation in vitro and in vivo.The human AML1a isoform encodes a truncated molecule with DNA-binding but no transactivation capacity. We used a retrovirus-based approach to transduce AML1a into primitive haematopoietic cells isolated from the mouse. We observed that enforced AML1a expression increased the competitive engraftment potential of murine long-term reconstituting stem cells with the proportion of AML1a-expressing cells increasing over time in both primary and secondary recipients. Furthermore, AML1a expression dramatically increased primitive and committed progenitor activity in engrafted animals as assessed by long-term culture, cobblestone formation, and colony assays. In contrast, expression of the full-length isoform AML1b abrogated engraftment potential. In vitro, AML1b promoted differentiation while AML1a promoted proliferation of progenitors capable of short-term lymphomyeloid engraftment. Consistent with these findings, the relative abundance of AML1a was highest in the primitive stem/progenitor compartment of human cord blood, and forced expression of AML1a in these cells enhanced maintenance of primitive potential both in vitro and in vivo.These data demonstrate that the "a" isoform of AML1 has the capacity to potentiate stem and progenitor cell engraftment, both of which are required for successful clinical transplantation. This activity is consistent with its expression pattern in both normal and leukaemic cells. Manipulating the balance of AML1 isoform expression may offer novel therapeutic strategies, exploitable in the contexts of leukaemia and also in cord blood

  5. Isoform-Specific Potentiation of Stem and Progenitor Cell Engraftment by AML1/RUNX1

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    Tsuzuki, Shinobu; Hong, Dengli; Gupta, Rajeev; Matsuo, Keitaro; Seto, Masao; Enver, Tariq

    2007-01-01

    Background AML1/RUNX1 is the most frequently mutated gene in leukaemia and is central to the normal biology of hematopoietic stem and progenitor cells. However, the role of different AML1 isoforms within these primitive compartments is unclear. Here we investigate whether altering relative expression of AML1 isoforms impacts the balance between cell self-renewal and differentiation in vitro and in vivo. Methods and Findings The human AML1a isoform encodes a truncated molecule with DNA-binding but no transactivation capacity. We used a retrovirus-based approach to transduce AML1a into primitive haematopoietic cells isolated from the mouse. We observed that enforced AML1a expression increased the competitive engraftment potential of murine long-term reconstituting stem cells with the proportion of AML1a-expressing cells increasing over time in both primary and secondary recipients. Furthermore, AML1a expression dramatically increased primitive and committed progenitor activity in engrafted animals as assessed by long-term culture, cobblestone formation, and colony assays. In contrast, expression of the full-length isoform AML1b abrogated engraftment potential. In vitro, AML1b promoted differentiation while AML1a promoted proliferation of progenitors capable of short-term lymphomyeloid engraftment. Consistent with these findings, the relative abundance of AML1a was highest in the primitive stem/progenitor compartment of human cord blood, and forced expression of AML1a in these cells enhanced maintenance of primitive potential both in vitro and in vivo. Conclusions These data demonstrate that the “a” isoform of AML1 has the capacity to potentiate stem and progenitor cell engraftment, both of which are required for successful clinical transplantation. This activity is consistent with its expression pattern in both normal and leukaemic cells. Manipulating the balance of AML1 isoform expression may offer novel therapeutic strategies, exploitable in the contexts of

  6. Cell line OCI/AML3 bears exon-12 NPM gene mutation-A and cytoplasmic expression of nucleophosmin.

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    Quentmeier, H; Martelli, M P; Dirks, W G; Bolli, N; Liso, A; Macleod, R A F; Nicoletti, I; Mannucci, R; Pucciarini, A; Bigerna, B; Martelli, M F; Mecucci, C; Drexler, H G; Falini, B

    2005-10-01

    We recently identified a new acute myeloid leukemia (AML) subtype characterized by mutations at exon-12 of the nucleophosmin (NPM) gene and aberrant cytoplasmic expression of NPM protein (NPMc+). NPMc+ AML accounts for about 35% of adult AML and it is associated with normal karyotype, wide morphological spectrum, CD34-negativity, high frequency of FLT3-ITD mutations and good response to induction therapy. In an attempt to identify a human cell line to serve as a model for the in vitro study of NPMc+ AML, we screened 79 myeloid cell lines for mutations at exon-12 of NPM. One of these cell lines, OCI/AML3, showed a TCTG duplication at exon-12 of NPM. This mutation corresponds to the type A, the NPM mutation most frequently observed in primary NPMc+ AML. OCI/AML3 cells also displayed typical phenotypic features of NPMc+ AML, that is, expression of macrophage markers and lack of CD34, and the immunocytochemical hallmark of this leukemia subtype, that is, the aberrant cytoplasmic expression of NPM. The OCI/AML3 cell line easily engrafts in NOD/SCID mice and maintains in the animals the typical features of NPMc+ AML, such as the NPM cytoplasmic expression. For all these reasons, the OCI/AML3 cell line represents a remarkable tool for biomolecular studies of NPMc+ AML.

  7. Transcription of the AML1/ETO chimera is guided by the P2 promoter of the AML1 gene in the Kasumi-1 cell line.

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    Markova, Elena N; Kantidze, Omar L; Razin, Sergey V

    2012-12-01

    Chromosomal translocation t (8;21)(q22;22) is one of the most frequent cytogenetic abnormalities found in acute myeloid leukaemia (AML). It generates the AML1/ETO fusion gene, which itself supports human haematopoietic stem cell self-renewal. However, the mechanism guiding transcription of this chimeric gene remains unclear. In our work, we attempted to shed light on this essential issue. We investigated the promoter from which transcription of the AML1/ETO gene is initiated and defined the three-dimensional structure of the whole rearranged locus. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Auger electron-emitting (111)In-DTPA-NLS-CSL360 radioimmunoconjugates are cytotoxic to human acute myeloid leukemia (AML) cells displaying the CD123(+)/CD131(-) phenotype of leukemia stem cells.

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    Gao, Catherine; Leyton, Jeffrey V; Schimmer, Aaron D; Minden, Mark; Reilly, Raymond M

    2016-04-01

    Chimeric IgG1 monoclonal antibody CSL360 recognizes the CD123(+)/CD131(-) phenotype expressed by leukemic stem cells (LSC). Auger electron-emitting (111)In-DTPA-NLS-CSL360 radioimmunoconjugates incorporating nuclear translocation sequence (NLS) peptides bound specifically to Raji cells transfected with CD123 and exhibited a KD of 11nmols/L in a competition receptor-binding assay using CD123-transfected CHO cells. (111)In-DTPA-NLS-CSL360 was bound, internalized and transported to the nucleus of human AML-5 myeloid leukemia cells. The clonogenic survival of AML-5 cells was reduced by (111)In-DTPA-NLS-CSL360 up to 3.7-fold. Isotype control (111)In-DTPA-chIgG1 was 2-fold less cytotoxic, and unlabeled CSL360, DTPA-NLS-CSL360 or free (111)In acetate did not decrease cell survival. These results are promising for further evaluation of (111)In-DTPA-NLS-CSL360 for Auger electron radioimmunotherapy of AML targeting the critical LSC subpopulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Identification of transcription factor AML-1 binding site upstream of human cytomegalovirus UL111A gene.

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    Xiaoqun Zheng

    Full Text Available Human cytomegalovirus (HCMV interleukin-10 (hcmvIL-10, encoded by HCMV UL111A gene, is a homolog of human IL-10. It exerts immunomodulatory effects that allow HCMV to evade host defense mechanisms. However, the exact mechanism underlying the regulation of hcmvIL-10 expression is not well understood. The transcription factor acute myeloid leukemia 1 (AML-1 plays an important role in the regulation of various genes involved in the differentiation of hematopoietic lineages. A putative AML-1 binding site is present within the upstream regulatory region (URR of UL111A gene. To provide evidence that AML-1 is involved in regulating UL111A gene expression, we examined the interaction of AML-1 with the URR of UL111A in HCMV-infected human monocytic THP-1 cells using a chromatin immunoprecipitation assay. HcmvIL-10 transcription was detected in differentiated THP-1 cells, but not in undifferentiated ones. Furthermore, the URR of UL111A showed a higher intensity of AML-1 binding, a higher level of histone H3 acetyl-K9, but a lower level of histone H3 dimethyl-K9 in differentiated THP-1 cells than undifferentiated cells. Down-regulation of AML1 by RNA interference decreased the expression of the UL111A gene. Our results suggest that AML-1 may contribute to the epigenetic regulation of UL111A gene via histone modification in HCMV-infected differentiated THP-1 cells. This finding could be useful for the development of new anti-viral therapies.

  10. Notch pathway activation targets AML-initiating cell homeostasis and differentiation.

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    Lobry, Camille; Ntziachristos, Panagiotis; Ndiaye-Lobry, Delphine; Oh, Philmo; Cimmino, Luisa; Zhu, Nan; Araldi, Elisa; Hu, Wenhuo; Freund, Jacquelyn; Abdel-Wahab, Omar; Ibrahim, Sherif; Skokos, Dimitris; Armstrong, Scott A; Levine, Ross L; Park, Christopher Y; Aifantis, Iannis

    2013-02-11

    Notch signaling pathway activation is known to contribute to the pathogenesis of a spectrum of human malignancies, including T cell leukemia. However, recent studies have implicated the Notch pathway as a tumor suppressor in myeloproliferative neoplasms and several solid tumors. Here we report a novel tumor suppressor role for Notch signaling in acute myeloid leukemia (AML) and demonstrate that Notch pathway activation could represent a therapeutic strategy in this disease. We show that Notch signaling is silenced in human AML samples, as well as in AML-initiating cells in an animal model of the disease. In vivo activation of Notch signaling using genetic Notch gain of function models or in vitro using synthetic Notch ligand induces rapid cell cycle arrest, differentiation, and apoptosis of AML-initiating cells. Moreover, we demonstrate that Notch inactivation cooperates in vivo with loss of the myeloid tumor suppressor Tet2 to induce AML-like disease. These data demonstrate a novel tumor suppressor role for Notch signaling in AML and elucidate the potential therapeutic use of Notch receptor agonists in the treatment of this devastating leukemia.

  11. KRAS (G12D Cooperates with AML1/ETO to Initiate a Mouse Model Mimicking Human Acute Myeloid Leukemia

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    Shanmin Zhao

    2014-01-01

    Full Text Available Background/Aims: It has been demonstrated that KRAS mutations represent about 90% of cancer-associated mutations, and that KRAS mutations play an essential role in neoplastic transformation. Cancer-associated RAS mutations occur frequently in acute myeloid leukemia (AML, suggesting a functional role for Ras in leukemogenesis. Methods: We successfully established a mouse model of human leukemia by transplanting bone marrow cells co-transfected with the K-ras (G12D mutation and AML1/ETO fusion protein. Results: Mice transplanted with AML/ETO+KRAS co-transduced cells had the highest mortality rate than mice transplanted with AML/ETO- or KRAS-transduced cells (115d vs. 150d. Upon reaching a terminal disease stage, EGFP-positive cells dominated their spleen, lymph nodes, peripheral blood and central nervous system tissue. Immunophenotyping, cytologic analyses revealed that AML/ETO+KRAS leukemias predominantly contained immature myeloid precursors (EGFP+/c-Kit+/Mac-1-/Gr-1-. Histologic analyses revealed that massive leukemic infiltrations were closely packed in dense sheets that effaced the normal architecture of spleen and thymus in mice transplanted with AML1/ETO + KRAS co-transduced cells. K-ras mRNA and protein expression were upregulated in bone marrow cells of the K-ras group and AML1/ETO + Kras group. The phosphorylation of MEK/ERK was significantly enhanced in the AML1/ETO + Kras group. The similar results of the AML1/ETO + Nras group were consistent with those reported previously. Conclusion: Co-transduction of KrasG12D and AML1/ETO induces acute monoblastic leukemia. Since expression of mutant K-ras alone was insufficient to induce leukemia, this model may be useful for investigating the multi-step leukemogenesis model of human leukemia.

  12. [Dendritic cells (DC) induced from acute myeloid leukemia (AML) cells with cytokine cocktails].

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    Yan, Kuang-hua; You, Sheng-guo; Bian, Shou-geng; Ma, Guan-jie; Ge, Wei; Ma, Shuang; Liu, Shi-he; Zhao, Chun-hua

    2003-07-01

    To explore the feasibility of DC being in vitro induced from AML cells with cytokine cocktails and their biological properties. AML cells were cultured in either presence or absence of cytokine cocktails. DC were studied for morphology, and cytochemical and immunofluorescent staining. Functions of DC were examined by MLC, FITC-conjugated dextran uptake test, and LDH release assay. RT-PCR and FISH were used to analyze the specific fusion genes of culture-derived DC. Classical DC morphological changes occurred in all 15 cultured AML cells. DC-associated surface molecules such as CD(1a), CD(80), CD(86), CD(106), CD(83) and HLA-DR were upregulated (P AML cells uncultured or cultured in the absence of cytokines (P CTL assay was performed in 5 of the 15 samples. At effector/target ratio of 20:1, auto-T lymphocytes primed with the culture-derived DC exhibited no more killing activity to auto-AML cells than those stimulated by IL-2 or uncultured AML cells. Culture-derived DC presenced the native AML-specific aberrant karyotype and related fusion gene. Cytokine cocktails could in vitro induce AML cells into DC with classical morphology, immunophenotype and function. DC maturity induced by different cytokine cocktails could be variable. Culture-derived DC were originated from the native AML cells. AML cells could make the auto-T lymphocyte anergy.

  13. Fusion of dendritic cells and CD34+CD38- acute myeloid leukemia (AML) cells potentiates targeting AML-initiating cells by specific CTL induction.

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    Lei, Zhang; Zhang, Gui-Mei; Hong, Mei; Feng, Zuo-Hua; Huang, Bo

    2009-05-01

    Distinct leukemia-initiating cells (L-ICs) represent a critical target for therapeutic intervention of acute myeloid leukemia (AML). A potential strategy to eradicate L-ICs is to generate L-IC-specific cytotoxic T lymphocytes (CTLs). However, owing to rarity and immortality of L-ICs, it is difficult for antigen-presenting cells to capture L-ICs for specific antigen presentation. Here, we report a novel approach by fusing allogeneic dendritic cells (DCs) and CD34CD38 AML progenitor cells, through which specific CTLs were effectively induced, leading to the cytolysis to AML-initiating cells. Fusion of either DC/CD34CD38 AML cell or DC/CD34 AML cell could effectively induce the proliferation and activation of CTLs. However, only the former CTLs could effectively attack AML progenitor cells, and result in the unkilled progenitor/initiating cells losing the abilities of active proliferation in vitro and engraftment in NOD-SCID mice. These findings suggest that AML progenitor/initiating cell-specific CTLs may be generated based on allogeneic DC/progenitor cell fusion strategy; the induced CTLs may potentially eradicate AML by targeting L-ICs directly or indirectly.

  14. ELMO1 is upregulated in AML CD34+ stem/progenitor cells, mediates chemotaxis and predicts poor prognosis in normal karyotype AML.

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    Marta E Capala

    Full Text Available Both normal as well leukemic hematopoietic stem cells critically depend on their microenvironment in the bone marrow for processes such as self-renewal, survival and differentiation, although the exact pathways that are involved remain poorly understood. We performed transcriptome analysis on primitive CD34+ acute myeloid leukemia (AML cells (n = 46, their more differentiated CD34- leukemic progeny, and normal CD34+ bone marrow cells (n = 31 and focused on differentially expressed genes involved in adhesion and migration. Thus, Engulfment and Motility protein 1 (ELMO1 was identified amongst the top 50 most differentially expressed genes. ELMO1 is a crucial link in the signaling cascade that leads to activation of RAC GTPases and cytoskeleton rearrangements. We confirmed increased ELMO1 expression at the mRNA and protein level in a panel of AML samples and showed that high ELMO1 expression is an independent negative prognostic factor in normal karyotype (NK AML in three large independent patient cohorts. Downmodulation of ELMO1 in human CB CD34+ cells did not significantly alter expansion, progenitor frequency or differentiation in stromal co-cultures, but did result in a decreased frequency of stem cells in LTC-IC assays. In BCR-ABL-transduced human CB CD34+ cells depletion of ELMO1 resulted in a mild decrease in proliferation, but replating capacity of progenitors was severely impaired. Downregulation of ELMO1 in a panel of primary CD34+ AML cells also resulted in reduced long-term growth in stromal co-cultures in two out of three cases. Pharmacological inhibition of the ELMO1 downstream target RAC resulted in a severely impaired proliferation and survival of leukemic cells. Finally, ELMO1 depletion caused a marked decrease in SDF1-induced chemotaxis of leukemic cells. Taken together, these data show that inhibiting the ELMO1-RAC axis might be an alternative way to target leukemic cells.

  15. Establishment of xenotransplantation model of human CN-AML with FLT3-ITD (mut) /NPM1 (-) in NOD/SCID mice.

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    Shang, Zhen; Wang, Jue; Wang, Di; Xiao, Min; Li, Tong-juan; Wang, Na; Huang, Liang; Zhou, Jian-feng

    2013-06-01

    Patients with FLT3-ITD (mut) /NPM1 (-) cytogenetically normal acute myeloid leukemia (CN-AML), as high-risk molecular group in CN-AML, are associated with a worse prognosis than other CN-AML patients. It is beneficial to generate xenotransplantation model of FLT3-ITD (mut) /NPM1 (-) CN-AML to better understand the pathogenesis and therapeutic strategies of such AML subtype. The purpose of present study was to establish the xenotransplantation model in NOD/SCID mice with FLT3-ITD (mut) /NPM1 (-) CN-AML primary cells. The FLT3-ITD (mut) /NPM1 (-) CN-AML primary cells from 3 of 7 cases were successfully transplanted into NOD/SCID mice, and human CD45 positive cells were detected in the peripheral blood, spleen and bone marrow of mice by using flow cytometry. Infiltration of human leukemia cells in various organs of mice was observed by using immunohistochemistry. Gene analysis confirmed sustained FLT3/ITD mutation without NPM1 mutation in mice. By performing serial transplantation, it was found that characteristics of the leukemia cells in secondary and tertiary generation models remained unchanged. Moreover, in vivo cytarabine administration could extend survival of NOD/SCID mice, which was consistent with clinical observation. In conclusion, we successfully established xenotransplantation model of human FLT3-ITD (mut) /NPM1 (-) CN-AML in NOD/SCID mice. The model was able to present primary disease and suitable to evaluate the curative effects of new drugs or therapy strategies.

  16. AKT signaling as a novel factor associated with in vitro resistance of human AML to gemtuzumab ozogamicin.

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    David B Rosen

    Full Text Available Gemtuzumab ozogamicin (GO, an immunoconjugate between an anti-CD33 antibody and a calicheamicin-γ(1 derivative, induces remissions and improves survival in a subset of patients with acute myeloid leukemia (AML. As the mechanisms underlying GO and calicheamicin-γ(1 resistance are incompletely understood, we herein used flow cytometry-based single cell network profiling (SCNP assays to study cellular responses of primary human AML cells to GO. Our data indicate that the extent of DNA damage is quantitatively impacted by CD33 expression and drug efflux activity. However, although DNA damage is required for GO-induced cytotoxicity, it is not sufficient for effective cell kill, suggesting that downstream anti-apoptotic pathways may function as relevant resistance mechanisms. Supporting this notion, we found activated PI3K/AKT signaling to be associated with GO resistance in vitro in primary AML cells. Consistently, the investigational AKT inhibitor MK-2206 significantly sensitized various human AML cells to GO or free calicheamicin-γ(1 with particularly pronounced effects in otherwise GO or free calicheamicin-γ(1-resistant cells. Likewise, MK-2206 also sensitized primary AML cells to calicheamicin-γ(1. Together, our findings illustrate the capacity of SCNP assays to discover chemotherapy-related biological pathways and signaling networks relevant to GO-induced genotoxic stress. The identification of AKT signaling as being associated with GO resistance in vitro may provide a novel approach to improve the in vivo efficacy of GO/calicheamicin-γ(1 and, by extrapolation, other DNA damage-based therapeutics.

  17. LIN28B Activation by PRL-3 Promotes Leukemogenesis and a Stem Cell-like Transcriptional Program in AML.

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    Zhou, Jianbiao; Chan, Zit-Liang; Bi, Chonglei; Lu, Xiao; Chong, Phyllis S Y; Chooi, Jing-Yuan; Cheong, Lip-Lee; Liu, Shaw-Cheng; Ching, Ying Qing; Zhou, Yafeng; Osato, Motomi; Tan, Tuan Zea; Ng, Chin Hin; Ng, Siok-Bian; Wang, Shi; Zeng, Qi; Chng, Wee-Joo

    2017-03-01

    PRL-3 (PTP4A3), a metastasis-associated phosphatase, is also upregulated in patients with acute myeloid leukemia (AML) and is associated with poor prognosis, but the underlying molecular mechanism is unknown. Here, constitutive expression of PRL-3 in human AML cells sustains leukemogenesis in vitro and in vivo Furthermore, PRL-3 phosphatase activity dependently upregulates LIN28B, a stem cell reprogramming factor, which in turn represses the let-7 mRNA family, inducing a stem cell-like transcriptional program. Notably, elevated levels of LIN28B protein independently associate with worse survival in AML patients. Thus, these results establish a novel signaling axis involving PRL-3/LIN28B/let-7, which confers stem cell-like properties to leukemia cells that is important for leukemogenesis.Implications: The current study offers a rationale for targeting PRL-3 as a therapeutic approach for a subset of AML patients with poor prognosis. Mol Cancer Res; 15(3); 294-303. ©2016 AACR.

  18. Augmentation of autologous T cell reactivity with acute myeloid leukemia (AML) blasts by Toll-like receptor (TLR) agonists

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    Zhong, Ruikun; Li, Hongying; Messer, Karen; Lane, Thomas A.; Zhou, Jiehua; Ball, Edward D.

    2015-01-01

    This study investigated whether TNF-α, Toll-like receptors (TLRs) 7/8 agonist resiquimod (R848), the TLR4 agonist lipopolysaccharide (LPS) and their combinations can enhance autologous AML-reactive T cell generation in an in vitro culture. AML peripheral blood or bone marrow mononuclear cells were cultured in medium supplemented with GM-CSF/IL-4 to induce dendritic cell (DC) differentiation of AML blasts (AML-DC). The impact of TNF-α, LPS, R848 and their combinations on AML-DC cultures was an...

  19. Novel agents inhibit human leukemic cells

    Institute of Scientific and Technical Information of China (English)

    Wei-ping YU; Juan LI

    2012-01-01

    Ouabain (OUA) and pyrithione zinc (PZ) have been proved as the potential drugs for treating acute myeloid leukemia (AML).Selected from a screening among 1040 Food and Drug Administration-approved pharmacological agents,both drugs showability to induce apoptosis of the culturing AML cells,exhibiting the poisoning effect on the cells.Studies also reveal the efficiency of the drugs in inhibiting the growth of human AML cells injected into the mice lacking of immunity and killing primary AML cells from the peripheral blood of AML patients[1].

  20. Acute myeloid leukemia (AML)-reactive cytotoxic T lymphocyte clones rapidly expanded from CD8(+) CD62L((high)+) T cells of healthy donors prevent AML engraftment in NOD/SCID IL2Rgamma(null) mice.

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    Distler, Eva; Wölfel, Catherine; Köhler, Sylvia; Nonn, Marion; Kaus, Nina; Schnürer, Elke; Meyer, Ralf G; Wehler, Thomas C; Huber, Christoph; Wölfel, Thomas; Hartwig, Udo F; Herr, Wolfgang

    2008-04-01

    Current in vitro techniques for isolating leukemia-reactive cytotoxic T lymphocytes (CTLs) from healthy donors are of relatively low efficiency and yield responder populations with unknown biological significance. This study aimed at the development of a more reliable approach, allowing generation and expansion of acute myeloid leukemia (AML)-reactive CTLs using primary in vitro stimulation. We established allogeneic mini-mixed lymphocyte-leukemia cultures (mini-MLLCs) by stimulating donor CD8(+) T cells with human leukocyte antigen (HLA) class I-matched AML blasts in microtiter plates. Before culture, CD8(+) T cells were separated into CD62L((high)+) and CD62L((low)+/neg) subsets enriched for naive/central memory and effector memory cells, respectively. In eight different related and unrelated donor/AML pairs, numerous CTL populations were isolated that specifically lysed myeloid leukemias in association with various HLA-A, -B, or -C alleles. These CTLs expressed T-cell receptors of single Vbeta-chain families, indicating their clonal origin. The majority of CTL clones were obtained from mini-MLLCs initiated with CD62L((high)+) cells. Using antigen-specific stimulation, multiple CTL populations were amplified to 10(8)-10(10) cells within 6 to 8 weeks. Three of four representative CTL clones were capable of completely preventing engraftment of human primary AML blasts in nonobese diabetic/severe combined immune deficient IL2Rgamma(null) mice. The mini-MLLC approach allows the efficient in vitro expansion of AML-reactive CTL clones from CD8(+)CD62L((high)+) precursors of healthy donors. These CTLs can inhibit leukemia engraftment in immunodeficient mice, suggesting their potential biological relevance.

  1. The Hematopoietic Transcription Factor AML1 (RUNX1) Is Negatively Regulated by the Cell Cycle Protein Cyclin D3

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    Peterson, Luke F.; Boyapati, Anita; Ranganathan, Velvizhi; Iwama, Atsushi; Tenen, Daniel G.; Tsai, Schickwann; Zhang, Dong-Er

    2005-01-01

    The family of cyclin D proteins plays a crucial role in the early events of the mammalian cell cycle. Recent studies have revealed the involvement of AML1 transactivation activity in promoting cell cycle progression through the induction of cyclin D proteins. This information in combination with our previous observation that a region in AML1 between amino acids 213 and 289 is important for its function led us to investigate prospective proteins associating with this region. We identified cyclin D3 by a yeast two-hybrid screen and detected AML1 interaction with the cyclin D family by both in vitro pull-down and in vivo coimmunoprecipitation assays. Furthermore, we demonstrate that cyclin D3 negatively regulates the transactivation activity of AML1 in a dose-dependent manner by competing with CBFβ for AML1 association, leading to a decreased binding affinity of AML1 for its target DNA sequence. AML1 and its fusion protein AML1-ETO have been shown to shorten and prolong the mammalian cell cycle, respectively. In addition, AML1 promotes myeloid cell differentiation. Thus, our observations suggest that the direct association of cyclin D3 with AML1 functions as a putative feedback mechanism to regulate cell cycle progression and differentiation. PMID:16287839

  2. CD14 mediated endogenous TNF-alpha release in HL60 AML cells: a potential model for CD14 mediated endogenous cytokine release in the treatment of AML.

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    Treon, S P; Anand, B; Ulevitch, R; Broitman, S A

    1994-01-01

    In previous studies, HL60 AML cells treated with tumor necrosis factor-alpha (TNF), interferon-gamma (IFN), and lipopolysaccharides (LPS) displayed decreased growth and viability, enhanced monocytic pathway differentiation and endogenous TNF release. Endogenous TNF release by LPS/TNF/IFN treated HL60 cells was postulated to play a role with the above findings. In these studies, HL60 cells expressed CD14 when treated with TNF, IFN, and LPS. CD14 mediates TNF release in monocytes/macrophages in response to binding of LPS with LPS binding protein (LBP). CD14 was not expressed in either untreated or LPS only treated HL60 cells. CD14 expression was present and greater with HL60 cells cultured with LPS/TNF/IFN vs TNF/IFN (47.47% vs 9.07% positive, respectively) suggesting synergism for LPS in CD14 induction. CD14 expression was associated with endogenous TNF release, and with significantly higher levels by HL60 cells treated with LPS/TNF/IFN vs TNF/IFN (p < 0.001). Addition of anti-CD14 antibody significantly reduced release of TNF in TNF/IFN (p < 0.001) and LPS/TNF/IFN (p = 0.0013) treated cells. KG1 and U937 AML cells treated with LPS, TNF, and IFN did not express CD14, nor release TNF. A model for inducing release of endogenous growth inhibitory cytokines by CD14 bearing AML cells is proposed as an approach to AML therapy.

  3. The effect of haemopoietic growth factors on the cell cycle of AML progenitors and their sensitivity to cytosine arabinoside in vitro.

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    Smith, M A; Singer, C R; Pallister, C J; Smith, J G

    1995-08-01

    This study examines the effect of pretreatment in liquid culture of acute myeloid leukaemic (AML) progenitors with recombinant human IL-3 or G and GM-CSF. Prior to and following cytokine priming, the sensitivity of cells to cytosine arabinoside (Ara-C) at concentrations ranging from 10(-12) to 10(-4) M was assessed in clonogenic culture. In addition, the initial percentage of AML cells in S phase was assessed and their subsequent kinetic response to cytokine treatment evaluated by FACScan analysis. Light-density marrow cells (LDMCs) from 19 AML patients were initially T-cell and monocyte depleted in order to remove potential sources of endogenous cytokine production prior to in vitro investigation. LDMCs were incubated in liquid phase for 7 d in a chemically defined complete medium with or without cytokines. Clonogenic data from fresh AML LDMCs not pretreated with growth factors demonstrated a heterogenous response to Ara-C. In only 4/15 marrows tested clonogenically was there any improvement in sensitivity to Ara-C following cytokine priming. S-phase data on all 19 marrows were similarly variable either before or after cytokine preincubation. There was no discernible correlation between clonogenic and kinetic data, nor could any relationship be established between in vitro findings and the FAB subtypes of patients or clinical outcomes. In summary, it would appear that the cell-cycle status of AML cells is likely to be only one of many contributory factors governing the sensitivity of AML progenitors to Ara-C. The clinical response of AML patients to cytokine therapy in association with cell-cycle-specific cytotoxic agents may therefore be variable and unpredictable.

  4. NK cell-mediated killing of AML blasts. Role of histamine, monocytes and reactive oxygen metabolites

    Energy Technology Data Exchange (ETDEWEB)

    Brune, M.; Mellqvist, U.H. [Sahlgren`s Univ. Hospital, Dept. of Medicine, Haematology Section, Goeteborg (Sweden); Hansson, M.; Hermodsson, S.; Hellstrand, K. [Sahlgren`s Univ. Hospital, Dept. of Virology, Goeteborg (Sweden)

    1996-10-01

    Blasts recovered from patients with acute myelogenous leukaemia (AML) were lysed by heterologeous natural killer (NK) cells treated with NK cell-activating cytokine-induced killing of AML blasts was inhibited by monocytes, recovered from peripheral blood by counterflow centrifugal elutriation. Histamine, at concentrations exceeding 0.1 {mu}M, abrogated the monocyte-induced inhibition of NK cells; thereby, histamine and IL-2 or histamine and IFN-{alpha} synergistically induced NK cell-mediated destruction of AML blasts. The effect of histamine was completely blocked by the histamine H2-receptor (H2R) antagonist ranitidine but not by its chemical control AH20399AA. Catalase, a scavenger of reactive oxygen metabolites (ROM), reversed the monocyte-induced inhibition of NK cell-mediated killing of blast cells, indicating that the inhibitory signal was mediated by products of the respiratory burst of monocytes. It is concluded that (i) monocytes inhibit anti-leukemic properties of NK cells, (ii) the inhibition is conveyed by monocyte-derived ROM, and (iii) histamine reverses the inhibitory signal and, thereby, synergizes with NK cell-activating cytokines to induce killing of AML blasts. (au) 19 refs.

  5. Inhibition of FLT3 expression by green tea catechins in FLT3 mutated-AML cells.

    Directory of Open Access Journals (Sweden)

    Bui Thi Kim Ly

    Full Text Available Acute myeloid leukemia (AML is a heterogeneous disease characterized by a block in differentiation and uncontrolled proliferation. FLT3 is a commonly mutated gene found in AML patients. In clinical trials, the presence of a FLT3-ITD mutation significantly correlates with an increased risk of relapse and dismal overall survival. Therefore, activated FLT3 is a promising molecular target for AML therapies. In this study, we have shown that green tea polyphenols including (--epigallocatechin-3-gallate (EGCG, (--epigallocatechin (EGC, and (--epicatechin-3-gallate (ECG suppress the proliferation of AML cells. Interestingly, EGCG, EGC and ECG showed the inhibition of FLT3 expression in cell lines harboring FLT3 mutations. In the THP-1 cells harboring FLT3 wild-type, EGCG showed the suppression of cell proliferation but did not suppress the expression of FLT3 even at the concentration that suppress 100% cell proliferation. Moreover, EGCG-, EGC-and ECG-treated cells showed the suppression of MAPK, AKT and STAT5 phosphorylation. Altogether, we suggest that green tea polyphenols could serve as reagents for treatment or prevention of leukemia harboring FLT3 mutations.

  6. Long-term survival after allogeneic haematopoietic cell transplantation for AML in remission

    DEFF Research Database (Denmark)

    Sengeløv, H; Gerds, T A; Brændstrup, P

    2013-01-01

    We report the results of non-myeloablative (NM) and myeloablative (MA) conditioning for haematopoietic cell transplantation in 207 consecutive AML patients at a single institution. A total of 122 patients were transplanted in first CR (CR1) and 67 in second CR (CR2). MA conditioning was given to ...

  7. [Reversed effect of valproic acid on transcription inhibition of AML1-ETO fusion protein of kasumi-1 leukemic cell line].

    Science.gov (United States)

    Zhao, Lei; Zhu, Cui-Min; Zhang, Zhi-Hua; Tian, Wen-Liang; Hao, Chang-Lai

    2009-04-01

    This study was aimed to investigate the mechanism of histone deacetylase (HDAC) inhibitor, valproic acid (VPA), reversing transcription inhibition of AML1-ETO fusion protein in Kasumi-1 cell line. The mRNA expressions of AML1-ETO, AML1 and cyclin D2 were detected by semi-quantitation RT-PCR after treating kasumi-1 cells with VPA at different doses/and different time points. The results indicated that the mRNA expression of AML1-ETO showed no obvious change, when kasumi-1 cells were treated with VPA. Compared with control group, the expression level of AML1 mRNA significantly increased in a dose-dependent manner. Compared with control group, the expression level of cyclin D2 mRNA significantly decreased when kasumi-1 cells had been treated with 3 mmol/L VPA as well as kasumi-1 cells were treated with different concentrations of VPA for 3 days. In conclusion, VPA could remove transcription inhibition of AML1-ETO fusion protein, increase transcription of AML1 and down-regulate mRNA expression of AML1 target gene cyclin D2 through HDAC inhibiting activity.

  8. Chimeric antigen receptor T cell therapy in AML: How close are we?

    Science.gov (United States)

    Gill, Saar

    2016-12-01

    The majority of patients presenting with acute myeloid leukemia (AML) initially respond to chemotherapy but post-remission therapy is required to consolidate this response and achieve long-term disease-free survival. The most effective form of post-remission therapy relies on T cell immunotherapy in the form of allogeneic hematopoietic cell transplantation (HCT). However, patients with active disease cannot usually expect to be cured with HCT. This inherent dichotomy implies that traditional T cell-based immunotherapy in the form of allogeneic HCT stops being efficacious somewhere between the measurable residual disease (MRD) and the morphologically obvious range. This is in part because the full power of T cells must be restrained in order to avoid lethal graft-versus-host disease (GVHD) and partly because only a sub-population of donor T cells are expected to be able to recognize AML cells via their T cell receptor. Chimeric antigen receptor (CAR) T cell therapy, most advanced in the treatment of patients with B-cell malignancies, may circumvent some of these limitations. However, major challenges remain to be overcome before CAR T cell therapy can be safely applied to AML.

  9. Chidamide, a novel histone deacetylase inhibitor, inhibits the viability of MDS and AML cells by suppressing JAK2/STAT3 signaling

    Science.gov (United States)

    Zhao, Sida; Guo, Juan; Zhao, Youshan; Fei, Chengming; Zheng, Qingqing; Li, Xiao; Chang, Chunkang

    2016-01-01

    Many studies have indicated that histone deacetylase (HDAC) activity is always increased in a lot of human tumors, and inhibition of HDAC activity is a promising new strategy in the treatment of cancers. Chidamide, a novel HDAC inhibitor of the benzamide class, is currently under clinical trials. In this study, we aimed to investigate the antitumor activity of Chidamide on myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) cell lines and explore the possible mechanism. Chidamide exhibited efficient anti-proliferative activity on MDS and AML cells in a time- and dose-dependent manner, accompanied by cell cycle arrest at G0/G1 phase and cell apoptosis. Importantly, Chidamide possessed potent HDAC inhibition property, as evaluated by HDAC activity analysis and acetylation of histone H3 and H4. Moreover, Chidamide significantly increased the expression of Suppressors of cytokine signaling 3 (SOCS3), reduced the expression of Janus activated kinases 2 (JAK2) and Signal transducer and activator of transcription 3 (STAT3), and inhibited STAT3 downstream genes, including c-Myc, Bcl-xL, and Mcl-1, which are involved in cell cycle progression and anti-apoptosis. Therefore, we demonstrate that Chidamide exhibits potent inhibitory effect on cell viability of MDS and AML cells, and the possible mechanism may lie in the downregulation of JAK2/STAT3 signaling through SOCS3 upregulation. Our data provide rationale for clinical investigations of Chidamide in MDS and AML. PMID:27508038

  10. Augmentation of autologous T cell reactivity with acute myeloid leukemia (AML) blasts by Toll-like receptor (TLR) agonists.

    Science.gov (United States)

    Zhong, RuiKun; Li, Hongying; Messer, Karen; Lane, Thomas A; Zhou, Jiehua; Ball, Edward D

    2015-06-01

    This study investigated whether TNF-α, Toll-like receptors (TLRs) 7/8 agonist resiquimod (R848), the TLR4 agonist lipopolysaccharide (LPS) and their combinations can enhance autologous AML-reactive T cell generation in an in vitro culture. AML peripheral blood or bone marrow mononuclear cells were cultured in medium supplemented with GM-CSF/IL-4 to induce dendritic cell (DC) differentiation of AML blasts (AML-DC). The impact of TNF-α, LPS, R848 and their combinations on AML-DC cultures was analyzed. Significantly enhanced CD80, CD40, CD83, CD54, HLA-DR and CD86 expression of AML cells was observed by addition of TNF-α, LPS, R848 alone or combinations. Induced CD80 expression of AML cells was significantly higher through the combination of TNF-α, LPS and R848 (T + L + R) than that by T alone. CTL induced from T + L + R, T + R, T + L, L + R and R, but not T, L alone stimulated cultures showed significantly higher IFN-γ release than the medium control in response to autologous AML cells. IFN-γ release by T + L + R was significantly higher than T or L alone, and T + R was significantly higher than T alone. CTL generated from T + L + R, T + L, T + R, L + R and L alone exerted significantly higher AML cell killing than medium control. AML cell killing by T + L + R and T + R was significantly higher than T or R alone. These results indicate that the combination of T + L + R induces a significantly enhanced antigen presentation effect of AML-DC. We speculate that the complementary effects of reagent combinations may better address the heterogeneity of responses to any single agent in AML cells from different patients.

  11. Regulation of Trib2 by an E2F1-C/EBPα feedback loop in AML cell proliferation.

    LENUS (Irish Health Repository)

    Rishi, Loveena

    2014-04-10

    The loss of regulation of cell proliferation is a key event in leukemic transformation, and the oncogene tribbles (Trib)2 is emerging as a pivotal target of transcription factors in acute leukemias. Deregulation of the transcription factor E2F1, normally repressed by CCAAT enhancer-binding protein α (C\\/EBPα)-p42, occurs in acute myeloid leukemia (AML), resulting in the perturbation of cell cycle and apoptosis, emphasizing its importance in the molecular pathogenesis of AML. Here we show that E2F family members directly regulate Trib2 in leukemic cells and identify a feedback regulatory loop for E2F1, C\\/EBPα, and Trib2 in AML cell proliferation and survival. Further analyses revealed that E2F1-mediated Trib2 expression was repressed by C\\/EBPα-p42, and in normal granulocyte\\/macrophage progenitor cells, we detect C\\/EBPα bound to the Trib2 promoter. Pharmacological inhibition of the cell cycle or Trib2 knockdown resulted in a block in AML cell proliferation. Our work proposes a novel paradigm whereby E2F1 plays a key role in the regulation of Trib2 expression important for AML cell proliferation control. Importantly, we identify the contribution of dysregulated C\\/EBPα and E2F1 to elevated Trib2 expression and leukemic cell survival, which likely contributes to the initiation and maintenance of AML and may have significant implications for normal and malignant hematopoiesis.

  12. YKL-40 in allogeneic hematopoietic cell transplantation after AML and myelodysplastic syndrome

    DEFF Research Database (Denmark)

    Kornblit, B; Wang, T; Lee, S J;

    2016-01-01

    YKL-40, also called chitinase-3-like-1 protein, is an inflammatory biomarker that has been associated with disease severity in inflammatory and malignant diseases, including AML, multiple myeloma and lymphomas. The objective of the current study was to assess the prognostic value of pretransplant......, otherwise equal, donors are available.Bone Marrow Transplantation advance online publication, 18 July 2016; doi:10.1038/bmt.2016.192....... recipient and donor plasma YKL-40 concentrations in patients with AML (n=624) or myelodysplastic syndrome (n=157) treated with allogeneic hematopoietic cell transplantation (HCT). In recipients, the plasma YKL-40 concentrations were increased when the HCT-comorbidity index was ⩾5 (P=0.028). There were...

  13. Optimizing outcomes following allogeneic hematopoietic progenitor cell transplantation in AML: the role of hypomethylating agents.

    Science.gov (United States)

    Martino, Massimo; Fedele, Roberta; Moscato, Tiziana; Ronco, Francesca

    2013-07-01

    Aberrant DNA methylation is a key pathological mechanism in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), and provides rationale for the clinical development of hypomethylating agents (HMAs) for the treatment of these diseases. One HMA, azacitidine (Vidaza®, Celgene Corp.), has demonstrated improved survival versus conventional care regimens in patients with intermediate-2/high-risk MDS and AML (20-30% blasts) and has a favorable tolerability profile. Emerging evidence indicates that azacitidine can have an immunomodulatory effect by, for example, increasing functional regulatory T-cell (Treg) numbers and killer-cell-immunoglobulin-like receptor expression. Allogeneic hematopoietic progenitor cell transplantation (allo HPCT) is the only potentially curative treatment approach in patients with advanced MDS or AML. Unfortunately, allo HPCT in these settings is limited because most patients are ineligible due to age/comorbidities, or are at a high risk of treatment failure due to disease relapse. Recent studies have shown that azacitidine after allo HPCT increases Treg numbers while inducing a cytotoxic CD8+ T-cell response, suggesting a potential mechanism for augmenting the graft-versus-leukemia (GvL) effect without increasing graft-versushost- disease (GVHD). In patients at a high risk of relapse following allo HPCT, pre-emptive azacitidine may help prevent/delay relapse. For patients who have relapsed following allo HPCT, azacitidine may be a salvage therapy option, either as monotherapy or in combination with donor lymphocyte infusions (DLI). In this mini-review, we discuss these emerging clinical data for HMAs in the post-allo HPCT regimens and highlight the possible future role of azacitidine in this setting.

  14. The NAE inhibitor pevonedistat interacts with the HDAC inhibitor belinostat to target AML cells by disrupting the DDR.

    Science.gov (United States)

    Zhou, Liang; Chen, Shuang; Zhang, Yu; Kmieciak, Maciej; Leng, Yun; Li, Lihong; Lin, Hui; Rizzo, Kathryn A; Dumur, Catherine I; Ferreira-Gonzalez, Andrea; Rahmani, Mohamed; Povirk, Lawrence; Chalasani, Sri; Berger, Allison J; Dai, Yun; Grant, Steven

    2016-05-05

    Two classes of novel agents, NEDD8-activating enzyme (NAE) and histone deacetylase (HDAC) inhibitors, have shown single-agent activity in acute myelogenous leukemia (AML)/myelodysplastic syndrome (MDS). Here we examined mechanisms underlying interactions between the NAE inhibitor pevonedistat (MLN4924) and the approved HDAC inhibitor belinostat in AML/MDS cells. MLN4924/belinostat coadministration synergistically induced AML cell apoptosis with or without p53 deficiency or FLT3-internal tandem duplication (ITD), whereas p53 short hairpin RNA (shRNA) knockdown or enforced FLT3-ITD expression significantly sensitized cells to the regimen. MLN4924 blocked belinostat-induced antiapoptotic gene expression through nuclear factor-κB inactivation. Each agent upregulated Bim, and Bim knockdown significantly attenuated apoptosis. Microarrays revealed distinct DNA damage response (DDR) genetic profiles between individual vs combined MLN4924/belinostat exposure. Whereas belinostat abrogated the MLN4924-activated intra-S checkpoint through Chk1 and Wee1 inhibition/downregulation, cotreatment downregulated multiple homologous recombination and nonhomologous end-joining repair proteins, triggering robust double-stranded breaks, chromatin pulverization, and apoptosis. Consistently, Chk1 or Wee1 shRNA knockdown significantly sensitized AML cells to MLN4924. MLN4924/belinostat displayed activity against primary AML or MDS cells, including those carrying next-generation sequencing-defined poor-prognostic cancer hotspot mutations, and CD34(+)/CD38(-)/CD123(+) populations, but not normal CD34(+) progenitors. Finally, combined treatment markedly reduced tumor burden and significantly prolonged animal survival (P DDR in AML/MDS cells. This strategy warrants further consideration in AML/MDS, particularly in disease with unfavorable genetic aberrations.

  15. Role of the Phosphorylation of mTOR in the Differentiation of AML Cells Triggered with CD44 Antigen

    KAUST Repository

    Darwish, Manar M

    2013-05-01

    Acute myeloid leukemia (AML) is a hematological disorder characterized by blockage of differentiation of myeloblasts. To date, the main therapy for AML is chemotherapy. Yet, studies are seeking a better treatment to enhance the survival rate of patients and minimize the relapsing of the disease. Since the major problem in these cells is that they are arrested in cellular differentiation, drugs that could induce their differentiation have proven to be efficient and of major interest for AML therapy. CD44 triggering appeared as a promising target for AML therapy as it has been shown that specific monoclonal antibodies, such as A3D8 and H90, reversed the blockage of differentiation, inhibited the proliferation of all AML subtypes, and in some cases, induced cell apoptosis. Studies conducted in our laboratory have added strength to these antibodies as potential treatment for AML. Indeed, our laboratory found that treating HL60 cells with A3D8 shows a decrease in the phosphorylation of the mammalian target of Rapamycin (mTOR) kinase correlated with the inhibition of proliferation/induction of differentiation of AML cells.The relationship between the induction of differentiation and the inhibition of proliferation and the decrease of mTOR phosphorylation remains to be clarified. To study the importance of the de-phosphorylation of mTOR and the observed effect of CD44 triggering on differentiation and/or proliferation, we sought to prepare phospho-mimic mutants of the mTOR kinase that will code for a constitutively phosphorylated form of mTOR and used two main methods to express this mutant in HL60 cells: lentiviral and simple transfection (cationic-liposomal transfection).

  16. In vitro generation of tumor specific T cells that recognize a shared antigen of AML: molecular characterization of TCR genes.

    Science.gov (United States)

    Coppage, Myra; Belanger, Todd; Zauderer, Maurice; Sahasrabudhe, Deepak

    2007-02-01

    The identification of immunologically relevant tumor antigens is hampered by the difficulty of generating tumor-specific cytotoxic T cells (CTL). We present data demonstrating in vitro induction of autologous acute myelogenous leukemia (AML)-specific CTL. The specific T cell receptor has been identified and cloned. The CTL demonstrated specific lysis to autologous tumor blasts, but not to autologous BLCL or the NK-sensitive target K562. The clone secreted GM-CSF, TNFa, and IFNg when stimulated with AML blasts from 3 of 11 patients or cell lines tested, but not with K562 or autologous B-LCL. These three AML samples share a single HLA Class I antigen, HLA-A24. The T cell receptor genes identified by molecular methods are Vbeta7.9-J2.3-Cbeta2 and Valpha17-J49-Calpha.

  17. Effects of TLR agonists on maturation and function of 3-day dendritic cells from AML patients in complete remission

    Directory of Open Access Journals (Sweden)

    Merk Martina

    2011-09-01

    Full Text Available Abstract Background Active dendritic cell (DC immunization protocols are rapidly gaining interest as therapeutic options in patients with acute myeloid leukemia (AML. Here we present for the first time a GMP-compliant 3-day protocol for generation of monocyte-derived DCs using different synthetic Toll-like receptor (TLR agonists in intensively pretreated patients with AML. Methods Four different maturation cocktails were compared for their impact on cell recovery, phenotype, cytokine secretion, migration, and lymphocyte activation in 20 AML patients and 25 healthy controls. Results Maturation cocktails containing the TLR7/8 agonists R848 or CL075, with and without the addition of the TLR3 agonist poly(I:C, induced DCs that had a positive costimulatory profile, secreted high levels of IL-12(p70, showed chemotaxis to CCR7 ligands, had the ability to activate NK cells, and efficiently stimulated antigen-specific CD8+ T cells. Conclusions Our results demonstrate that this approach translates into biologically improved DCs, not only in healthy controls but also in AML patients. This data supports the clinical application of TLR-matured DCs in patients with AML for activation of innate and adaptive immune responses.

  18. Donor-Derived Smoldering Multiple Myeloma following a Hematopoietic Cell Transplantation for AML

    Science.gov (United States)

    Fiala, Mark; Slade, Michael; Westervelt, Peter

    2017-01-01

    Posttransplant Lymphoproliferative Disorder (PTLD) is one of the most common malignancies complicating solid organ transplantation. In contrast, PTLD accounts for a minority of secondary cancers following allogeneic hematopoietic cell transplantation (HCT). Here we report on a 61-year-old woman who received an ABO-mismatched, HLA-matched unrelated donor hematopoietic cell transplantation from a presumably healthy donor for a diagnosis of acute myeloid leukemia (AML). Eighteen months following her transplant, she developed a monoclonal gammopathy. Bone marrow studies revealed 10% plasma cells, but the patient lacked clinical defining features of multiple myeloma (MM); thus a diagnosis of smoldering multiple myeloma (SMM) was established. Cytogenetic and molecular studies of the bone marrow confirmed the plasma cells were donor-derived. The donor lacks a diagnosis of monoclonal gammopathy of undetermined significance, SMM, or MM. PMID:28316846

  19. Donor-Derived Smoldering Multiple Myeloma following a Hematopoietic Cell Transplantation for AML

    Directory of Open Access Journals (Sweden)

    Bita Fakhri

    2017-01-01

    Full Text Available Posttransplant Lymphoproliferative Disorder (PTLD is one of the most common malignancies complicating solid organ transplantation. In contrast, PTLD accounts for a minority of secondary cancers following allogeneic hematopoietic cell transplantation (HCT. Here we report on a 61-year-old woman who received an ABO-mismatched, HLA-matched unrelated donor hematopoietic cell transplantation from a presumably healthy donor for a diagnosis of acute myeloid leukemia (AML. Eighteen months following her transplant, she developed a monoclonal gammopathy. Bone marrow studies revealed 10% plasma cells, but the patient lacked clinical defining features of multiple myeloma (MM; thus a diagnosis of smoldering multiple myeloma (SMM was established. Cytogenetic and molecular studies of the bone marrow confirmed the plasma cells were donor-derived. The donor lacks a diagnosis of monoclonal gammopathy of undetermined significance, SMM, or MM.

  20. Cytotoxic T cell response against the chimeric ETV6-AML1 protein in childhood acute lymphoblastic leukemia.

    Science.gov (United States)

    Yotnda, P; Garcia, F; Peuchmaur, M; Grandchamp, B; Duval, M; Lemonnier, F; Vilmer, E; Langlade-Demoyen, P

    1998-07-15

    Cytotoxic T lymphocytes (CTL) are potent effector cells that could provide long term antitumor immunity if induced by appropriate vaccines. CTL recognize 8-14 amino acid-long peptides processed intracellularly and presented by MHC class I molecules. A well-characterized example of a potential tumor antigen in childhood pre-B Acute Lymphoblastic Leukemia (ALL) results from the chromosomal translocation 12;21 leading to the fusion of the ETV6 and AML1 genes. This translocation is observed in > 25% of ALL-patients. In this study, we have examined whether the chimeric ETV6-AML1 protein could serve as a tumor specific antigen for CTL in HLA-A2.1 individuals. We have identified a nonapeptide (RIAECILGM), encoded by the fusion region of the ETV6-AML1 protein, that binds to HLA-A2.1 molecules and induces specific primary CTL in peripheral blood lymphocytes from healthy donors. These CTL specifically lysed HLA-A2.1 tumor cells endogeneously expressing the ETV6-AML fusion protein. CTL with similar functional capacities were found with high frequencies and cloned from one patient's bone marrow indicating that ETV6-AML1-specific anti-ALL CTL are, at least in some patients, spontaneously stimulated and might participate to host antileukemia defense.

  1. High-affinity FRβ-specific CAR T cells eradicate AML and normal myeloid lineage without HSC toxicity.

    Science.gov (United States)

    Lynn, R C; Feng, Y; Schutsky, K; Poussin, M; Kalota, A; Dimitrov, D S; Powell, D J

    2016-06-01

    Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here we isolated a high-affinity (HA) folate receptor beta (FRβ)-specific single-chain variable fragment (2.48 nm KD) for optimization of FRβ-redirected CAR T-cell therapy for AML. T cells stably expressing the HA-FRβ CAR exhibited greatly enhanced antitumor activity against FRβ(+) AML in vitro and in vivo compared with a low-affinity FRβ CAR (54.3 nm KD). Using the HA-FRβ immunoglobulin G, FRβ expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34(+) hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRβ CAR T cells lysed mature CD14(+) monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRβ CAR T cells retained effective antitumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRβ CAR T cells is highly effective against AML and reduces the risk for long-term myeloid toxicity.

  2. Esculetin Downregulates the Expression of AML1-ETO and C-Kit in Kasumi-1 Cell Line by Decreasing Half-Life of mRNA.

    Science.gov (United States)

    Sawney, Sharad; Arora, Rashi; Aggarwal, Kamal K; Saluja, Daman

    2015-01-01

    One of the most frequent genetic aberrations in acute myeloid leukemia (AML) is chromosomal translocation between AML1/RUNX1 on chromosome 21 and ETO gene on chromosome 8 resulting in the expression of chimeric oncogene AML1-ETO. Although patients with t(8;21) translocation have good prognosis, 5-year survival is observed only in 50% of the cases. AML1-ETO translocation is usually accompanied by overexpression of mutant C-Kit, a tyrosine kinase, which contributes to uncontrolled proliferation of premature blood cells leading to relapse and poor prognosis. We illustrate the potential use of esculetin on leukemic cell line, Kasumi-1, bearing t(8;21) translocation and mutated C-Kit gene. Esculetin decreases the expression of AML1-ETO at both protein and transcript level within 24 hours of treatment. Half-life of AML1-ETO mRNA was reduced from 7 hours to 1.5 hours. Similarly half-life of C-Kit mRNA was reduced to 2 hours from 5 hours in esculetin treated cells. Esculetin also perturbed the expression of ectopically expressed AML1-ETO in U937 cells. The decreased expression of AML1-ETO chimeric gene was associated with increased expression of LAT1 and RUNX3 genes, targets of AML1. We envisage that discovery of a drug candidate which could target both these mutated genes would be a considerable breakthrough for future application.

  3. Esculetin Downregulates the Expression of AML1-ETO and C-Kit in Kasumi-1 Cell Line by Decreasing Half-Life of mRNA

    Directory of Open Access Journals (Sweden)

    Sharad Sawney

    2015-01-01

    Full Text Available One of the most frequent genetic aberrations in acute myeloid leukemia (AML is chromosomal translocation between AML1/RUNX1 on chromosome 21 and ETO gene on chromosome 8 resulting in the expression of chimeric oncogene AML1-ETO. Although patients with t(8;21 translocation have good prognosis, 5-year survival is observed only in 50% of the cases. AML1-ETO translocation is usually accompanied by overexpression of mutant C-Kit, a tyrosine kinase, which contributes to uncontrolled proliferation of premature blood cells leading to relapse and poor prognosis. We illustrate the potential use of esculetin on leukemic cell line, Kasumi-1, bearing t(8;21 translocation and mutated C-Kit gene. Esculetin decreases the expression of AML1-ETO at both protein and transcript level within 24 hours of treatment. Half-life of AML1-ETO mRNA was reduced from 7 hours to 1.5 hours. Similarly half-life of C-Kit mRNA was reduced to 2 hours from 5 hours in esculetin treated cells. Esculetin also perturbed the expression of ectopically expressed AML1-ETO in U937 cells. The decreased expression of AML1-ETO chimeric gene was associated with increased expression of LAT1 and RUNX3 genes, targets of AML1. We envisage that discovery of a drug candidate which could target both these mutated genes would be a considerable breakthrough for future application.

  4. Changes in the MALT1-A20-NF-κB expression pattern may be related to T cell dysfunction in AML.

    Science.gov (United States)

    Shi, Li; Chen, Shaohua; Lu, Yuhong; Wang, Xu; Xu, Ling; Zhang, Fan; Yang, Lijian; Wu, Xiuli; Li, Bo; Li, Yangqiu

    2013-04-30

    To elucidate the characteristics of T-cell receptor (TCR) signal transduction in T-cells from acute myeloid leukemia (AML), the mucosa-associated-lymphoid-tissue lymphoma-translocation gene 1 (MALT1), A20, NF-κB and MALT1-V1 gene expression levels in CD3+ T cells sorted from the peripheral blood of patients with AML were analyzed by real-time PCR. A significantly lower MALT1 and A20 expression level was found in T cells from patients with AML compared with healthy controls (p = 0.045, p MALT1-V1 (variant 1) was significantly higher in the AML group than in the healthy control group (p = 0.006), and the expression level of NF-κB was increased in the AML group. In conclusion, the characteristics of the expression pattern of MALT1-A20-NF-κB and the distribution of MALT1 variants in T cells from AML were first characterized. Overall, low TCR-CD3 signaling is related to low MALT1 expression, which may related to T cell immunodeficiency, while the up-regulation of MALT1-V1 may play a role in overcoming the T cell activity by downregulating A20 in patients with AML, which may be related to a specific response to AML-associated antigens.

  5. Detailed Characterization of Mesenchymal Stem/Stromal Cells from a Large Cohort of AML Patients Demonstrates a Definitive Link to Treatment Outcomes

    Directory of Open Access Journals (Sweden)

    Rafael Diaz de la Guardia

    2017-06-01

    Full Text Available Bone marrow mesenchymal stem/stromal cells (BM-MSCs are key components of the hematopoietic niche thought to have a direct role in leukemia pathogenesis. BM-MSCs from patients with acute myeloid leukemia (AML have been poorly characterized due to disease heterogeneity. We report a functional, genetic, and immunological characterization of BM-MSC cultures from 46 AML patients, stratified by molecular/cytogenetics into low-risk (LR, intermediate-risk (IR, and high-risk (HR subgroups. Stable MSC cultures were successfully established and characterized from 40 of 46 AML patients irrespective of the risk subgroup. AML-derived BM-MSCs never harbored tumor-specific cytogenetic/molecular alterations present in blasts, but displayed higher clonogenic potential than healthy donor (HD-derived BM-MSCs. Although HD- and AML-derived BM-MSCs equally provided chemoprotection to AML cells in vitro, AML-derived BM-MSCs were more immunosuppressive/anti-inflammatory, enhanced suppression of lymphocyte proliferation, and diminished secretion of pro-inflammatory cytokines. Multivariate analysis revealed that the level of interleukin-10 produced by AML-derived BM-MSCs as an independent prognostic factor negatively affected overall survival. Collectively our data show that AML-derived BM-MSCs are not tumor related, but display functional differences contributing to therapy resistance and disease evolution.

  6. Treatment, risk factors, and outcome of adults with relapsed AML after reduced intensity conditioning for allogeneic stem cell transplantation

    NARCIS (Netherlands)

    C. Schmid (Christoph); M. Labopin (Myriam); A. Nagler (Arnon); D. Niederwieser (Dietger); L. Castagna (Luca); R. Tabrizi (Reza); M. Stadler (Michael); J. Kuball (Jürgen); J.J. Cornelissen (Jan); J. Vorlicek (Jiri); G. Socié (Gerard); M. Falda (M.); L. Vindelov (Lars); P. Ljungman (Per); G. Jackson (Graham); N. Kröger; A. Rank (Andreas); E. Polge (Emmanuelle); V. Rocha (Vanderson); M. Mohty (Mohamad)

    2012-01-01

    textabstractBecause information on management and outcome of AML relapse after allogeneic hematopoietic stem cell transplantation (HSCT) with reduced intensity conditioning (RIC) is scarce, a retrospective registry study was performed by the Acute LeukemiaWorking Party of EBMT. Among 2815 RIC

  7. Treatment, risk factors, and outcome of adults with relapsed AML after reduced intensity conditioning for allogeneic stem cell transplantation

    DEFF Research Database (Denmark)

    Schmid, Christoph; Labopin, Myriam; Nagler, Arnon

    2012-01-01

    Since information on management and outcome of adults with AML relapsing after allogeneic hematopoietic stem cell transplantation with reduced intensity conditioning (RIC HSCT) is scarce, a retrospective registry study was performed by the Acute Leukemia Working Party of EBMT. Among 2815 RIC tran...

  8. Treosulfan, Fludarabine and 2 Gy Total Body Irradiation Followed by Allogeneic Hematopoietic Cell Transplantation in Patients with MDS and AML

    Science.gov (United States)

    Gyurkocza, Boglarka; Gutman, Jonathan; Nemecek, Eneida R.; Bar, Merav; Milano, Filippo; Ramakrishnan, Aravind; Scott, Bart; Fang, Min; Wood, Brent; Pagel, John M.; Baumgart, Joachim; Delaney, Colleen; Maziarz, Richard T.; Sandmaier, Brenda M.; Estey, Elihu H.; Appelbaum, Frederick R.; Storer, Barry E.; Deeg, H. Joachim

    2014-01-01

    Allogeneic hematopoietic cell transplantation (HCT) offers curative therapy for many patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). However, post-HCT relapse remains a major problem, particularly in patients with high-risk cytogenetics. In this prospective phase II trial we assessed the efficacy and toxicity of treosulfan, fludarabine and 2 Gy total body irradiation (TBI) as conditioning for allogeneic HCT in patients with MDS or AML. Ninety-six patients with MDS (n=36; 15 RMCD; 10 RAEB-1; 10 RAEB-2; 1 CMML-1) or AML (n=60; 35 CR1; 18 CR2; 3 advanced CR; 4 refractory relapse) were enrolled; median age was 51 (range: 1–60) years. Twelve patients had undergone a prior HCT with high intensity conditioning. Patients received intravenous (IV) treosulfan, 14 g/m2/day on days −6 to −4, IV fludarabine, 30 mg/m2/day on days −6 to −2, and 2 Gy TBI on day 0, followed by infusion of hematopoietic cells from related (n=27) or unrelated (n=69) donors. Graft-vs.-host disease prophylaxis consisted of tacrolimus and methotrexate. With a median follow-up of 30 months, the 2-year overall survival (OS), relapse incidence and non-relapse mortality were 73%, 27% and 8%, respectively. The incidences of grades II–IV (III–IV) acute and chronic graft-versus-host disease were 59% (10%) and 47%, respectively. Two-year OS was not significantly different between MDS patients with poor risk and good/intermediate risk cytogenetics (69% and 85%, respectively), or between AML patients with unfavorable and favorable/intermediate risk cytogenetics (64% and 76%, respectively). In AML patients, minimal residual disease (MRD; n=10) at the time of HCT predicted higher relapse incidence (70% vs. 18%) and lower OS (41% vs. 79%) at 2 years, when compared to patients without MRD. In conclusion, treosulfan, fludarabine and low-dose TBI provided effective conditioning for allogeneic HCT in patients with MDS or AML, and resulted in low relapse incidence, regardless

  9. Induction of MTG8-specific cytotoxic T-cell lines: MTG8 is probably a tumour antigen that is recognized by cytotoxic T cells in AML1-MTG8-fused gene-positive acute myelogenous leukaemia.

    Science.gov (United States)

    Maeda, M; Otsuka, T; Kimura, N; Kozu, T; Fukuyama, T; Uchida, N; Sugio, Y; Itoh, Y; Iino, T; Inaba, S; Niho, Y

    2000-11-01

    Several reports have demonstrated the persistent detection of AML1-MTG8 fusion products, representing minimal residual disease (MRD), in patients with t(8;21) acute myelogenous leukaemia (AML) who are in long-term remission. It is probable that immune-mediated mechanisms that are able to suppress the expansion of MRD may result in the continuance of remission. It was previously shown that some t(8;21) AML patients had high anti-MTG8 antibody titres. MTG8 expression in normal adult tissues is limited to the brain or heart in which human leucocyte antigen (HLA) class I cell-surface antigens are either not or are only faintly detectable. We hypothesized that the overexpression of the MTG8 gene in t(8;21) AML cells could act as a possible tumour antigen, which might be able to induce the immune-mediated suppression of the expansion of MRD. We were able to induce HLA-A0201-restricted cytotoxic T-lymphocyte (CTL) lines against an MTG8 peptide (MTG8b amino acids 182-191) using monocyte-derived dendritic cells from a healthy donor. T-cell receptor (TCR)Valpha17, TCRVbeta14 and 15, and TCRJbeta2.1 and 2.3 are predominantly used in these CTL lines. Our data, which suggest that the MTG8 protein could be one of the tumour antigens recognized by CTLs, may be helpful in further investigations of TCR analysis in t(8;21) AML patients with HLA-A0201 who are in long-term remission.

  10. NCAM(CD56) and RUNX1(AML1) Are Up-Regulated in Human Ischemic Cardiomyopathy and a Rat Model of Chronic Cardiac Ischemia

    Science.gov (United States)

    Gattenlöhner, Stefan; Waller, Christiane; Ertl, Georg; Bültmann, Burkhard-Dieter; Müller-Hermelink, Hans-Konrad; Marx, Alexander

    2003-01-01

    Chronic myocardial ischemia is the leading cause of impaired myocardial contractility and heart failure. To identify differentially expressed genes in human ischemic cardiomyopathy (ICM), we constructed a subtracted cDNA library using specimens of ICM compared to normal human heart. Among 100 randomly sequenced clones, seven sequences represented recently identified candidate genes for differential expression in cardiac hypertrophy. A further clone without a known hypertrophy-association coded for the adhesion molecule NCAM(CD56). RNase protection assay, immunohistochemistry, and Western blotting revealed strong overexpression of NCAM(CD56) in all hearts with ICM (n = 14) compared to normal hearts (n = 8), whereas in congestive cardiomyopathy (CCM) (n = 8), hypertrophic obstructive cardiomyopathy (n = 2), myocarditis (n = 4), and sarcoidosis (n = 2), at most slight overexpression of NCAM(CD56) was observed. NCAM(CD56) overexpression abnormally involved the whole cell membrane and the cytoplasma of cardiomyocytes only inside and adjacent to ischemia-induced cardiac scars. Normal or hypertrophic fibers at a distance from ischemic scars were devoid of NCAM overexpression. Identical alterations were observed in an experimental rat ICM model, but not in normal nor in spontaneously hypertensive rat hearts. In search of NCAM(CD56)-related transcription factors we found RUNX1(AML1) up-regulation in ICM and detected RUNX1(AML1) binding within the NCAM(CD56) promoter by electromobility shift assay. We concluded that strong overexpression of NCAM(CD56) and RUNX1(AML1) is a constant and characteristic feature of cardiomyocytes within or adjacent to scars in ICM. PMID:12937148

  11. Mapping of the mouse homolog of the human runt domain gene, AML2, to the distal region of mouse chromosome 4

    Energy Technology Data Exchange (ETDEWEB)

    Avraham, K.B.; Copeland, N.G.; Jenkins, N.A. [National Cancer Institute-Frederick Cancer Research and Development Center, MD (United States)] [and others

    1995-01-20

    AML2 is a runt domain belonging to a group of transcription factors that appear to play a role in Drosophila embryogenesis and mammalian oncogenic transformation. AML2 maps to human chromosome 1p36, a region involved in the t(1;3)(p36;q21) translocation found in association with myelodysplastic syndrome (MDS), myeloproliferative disease (MPD), and acute nonlymphocytic leukemia. 9 refs., 1 fig.

  12. Effect of the histone deacetylase inhibitor depsipeptide on B-cell differentiation in both TEL-AML1-positive and negative childhood acute lymphoblastic leukemia.

    Science.gov (United States)

    Stams, Wendy A G; den Boer, Monique L; Beverloo, H Berna; Kazemier, Karin M; van Wering, Elisabeth R; Janka-Schaub, Gritta E; Pieters, Rob

    2005-12-01

    The fusion protein TEL-AML1 in t(12;21)+ acute lymphoblastic leukemia (ALL) recruits co-repressors and histone deacetylases (HDAC), which transrepress AML1 target genes. Normal bone marrow cells were more resistant to HDAC inhibitor FK228 induced cell killing than were cells from ALL patients with or without t(12;21). FK228 induced differentiation in ALL, irrespective of the presence of t(12;21).

  13. Activity of a selective inhibitor of nuclear export, selinexor (KPT-330), against AML-initiating cells engrafted into immunosuppressed NSG mice.

    Science.gov (United States)

    Etchin, J; Montero, J; Berezovskaya, A; Le, B T; Kentsis, A; Christie, A L; Conway, A S; Chen, W C; Reed, C; Mansour, M R; Ng, C E L; Adamia, S; Rodig, S J; Galinsky, I A; Stone, R M; Klebanov, B; Landesman, Y; Kauffman, M; Shacham, S; Kung, A L; Wang, J C Y; Letai, A; Look, A T

    2016-01-01

    Currently available combination chemotherapy for acute myeloid leukemia (AML) often fails to result in long-term remissions, emphasizing the need for novel therapeutic strategies. We reasoned that targeted inhibition of a prominent nuclear exporter, XPO1/CRM1, could eradicate self-renewing leukemia-initiating cells (LICs) whose survival depends on timely XPO1-mediated transport of specific protein and RNA cargoes. Using an immunosuppressed mouse model bearing primary patient-derived AML cells, we demonstrate that selinexor (KPT-330), an oral antagonist of XPO1 that is currently in clinical trials, has strong activity against primary AML cells while sparing normal stem and progenitor cells. Importantly, limiting dilution transplantation assays showed that this cytotoxic activity is not limited to the rapidly proliferating bulk population of leukemic cells but extends to the LICs, whose inherent drug resistance and unrestricted self-renewal capacity has been implicated in the difficulty of curing AML patients with conventional chemotherapy alone.

  14. Cannabidiol stimulates Aml-1a-dependent glial differentiation and inhibits glioma stem-like cells proliferation by inducing autophagy in a TRPV2-dependent manner.

    Science.gov (United States)

    Nabissi, Massimo; Morelli, Maria Beatrice; Amantini, Consuelo; Liberati, Sonia; Santoni, Matteo; Ricci-Vitiani, Lucia; Pallini, Roberto; Santoni, Giorgio

    2015-10-15

    Glioma stem-like cells (GSCs) correspond to a tumor cell subpopulation, involved in glioblastoma multiforme (GBM) tumor initiation and acquired chemoresistance. Currently, drug-induced differentiation is considered as a promising approach to eradicate this tumor-driving cell population. Recently, the effect of cannabinoids (CBs) in promoting glial differentiation and inhibiting gliomagenesis has been evidenced. Herein, we demonstrated that cannabidiol (CBD) by activating transient receptor potential vanilloid-2 (TRPV2) triggers GSCs differentiation activating the autophagic process and inhibits GSCs proliferation and clonogenic capability. Above all, CBD and carmustine (BCNU) in combination overcome the high resistance of GSCs to BCNU treatment, by inducing apoptotic cell death. Acute myeloid leukemia (Aml-1) transcription factors play a pivotal role in GBM proliferation and differentiation and it is known that Aml-1 control the expression of several nociceptive receptors. So, we evaluated the expression levels of Aml-1 spliced variants (Aml-1a, b and c) in GSCs and during their differentiation. We found that Aml-1a is upregulated during GSCs differentiation, and its downregulation restores a stem cell phenotype in differentiated GSCs. Since it was demonstrated that CBD induces also TRPV2 expression and that TRPV2 is involved in GSCs differentiation, we evaluated if Aml-1a interacted directly with TRPV2 promoters. Herein, we found that Aml-1a binds TRPV2 promoters and that Aml-1a expression is upregulated by CBD treatment, in a TRPV2 and PI3K/AKT dependent manner. Altogether, these results support a novel mechanism by which CBD inducing TRPV2-dependent autophagic process stimulates Aml-1a-dependent GSCs differentiation, abrogating the BCNU chemoresistance in GSCs.

  15. CD8(+)T cells expressing both PD-1 and TIGIT but not CD226 are dysfunctional in acute myeloid leukemia (AML) patients.

    Science.gov (United States)

    Wang, Mengjie; Bu, Jin; Zhou, Maohua; Sido, Jessica; Lin, Yu; Liu, Guanfang; Lin, Qiwen; Xu, Xiuzhang; Leavenworth, Jianmei W; Shen, Erxia

    2017-09-08

    Acute myeloid leukemia (AML) is one of the most common types of leukemia among adults with an overall poor prognosis and very limited treatment management. Immune checkpoint blockade of PD-1 alone or combined with other immune checkpoint blockade has gained impressive results in murine AML models by improving anti-leukemia CD8(+)T cell function, which has greatly promoted the strategy to utilize combined immune checkpoint inhibitors to treat AML patients. However, the expression profiles of these inhibitory receptors in T cells from AML patients have not been clearly defined. Here we have defined subsets of CD8(+) and CD4(+) T cells in the peripheral blood (PB) from newly diagnosed AML patients and healthy controls (HCs). We have observed increased frequencies of PD-1- and TIGIT expressing CD8(+) T cells but decreased occurrence of CD226-expressing CD8(+)T cells in AML patients. Further analysis of these CD8(+) T cells revealed a unique CD8(+) T cell subset that expressed PD-1 and TIGIT but displayed lower levels of CD226 was associated with failure to achieve remission after induction chemotherapy and FLT3-ITD mutations which predict poor clinical prognosis in AML patients. Importantly, these PD-1(+)TIGIT(+)CD226(-)CD8(+)T cells are dysfunctional with lower expression of intracellular IFN-γ and TNF-α than their counterparts in HCs. Therefore, our studies revealed that an increased frequency of a unique CD8(+) T cell subset, PD-1(+)TIGIT(+)CD226(-)CD8(+) T cells, is associated with CD8(+)T cell dysfunction and poor clinical prognosis of AML patients, which may reveal critical diagnostic or prognostic biomarkers and direct more efficient therapeutic strategies. Copyright © 2017. Published by Elsevier Inc.

  16. Granulocytic Sarcoma by AML M4eo (inv16 after Allogeneic Stem Cell Transplantation without Bone Marrow Involvement

    Directory of Open Access Journals (Sweden)

    Stephan Zaenker

    2011-01-01

    Full Text Available Granulocytic sarcoma (GS represents a rare type of extramedullar manifestation from the acute myeloid leukaemia (AML. We report the case of a patient with recurrences of AML M4eo leukaemia in the uterus and the small intestine at 3 and 5 years, respectively, after matched related peripheral blood stem cell transplantation (PBSCT. The patient underwent the withdrawal of immunosuppression, hysterectomy, and local irradiation at first relapse, as well as systemic chemotherapy and donor lymphocyte infusions at second recurrence, inducing a second and third complete remission, respectively. At year six after transplantation, the patient experienced disease progression by meningeosis leukaemia to which she succumbed despite intrathecal chemotherapy. Following allogeneic stem cell transplantation, awareness for atypical manifestations of granulocytic sarcoma appears prudent, the cellular immunotherapy should aim at immunological disease control.

  17. Matrine induces apoptosis in human acute myeloid leukemia cells via the mitochondrial pathway and Akt inactivation.

    Directory of Open Access Journals (Sweden)

    Shenghui Zhang

    Full Text Available Acute myeloid leukemia (AML is a hematological malignancy characterized by a rapid increase in the number of immature myeloid cells in bone marrow. Despite recent advances in the treatment, AML remains an incurable disease. Matrine, a major component extracted from Sophora flavescens Ait, has been demonstrated to exert anticancer effects on various cancer cell lines. However, the effects of matrine on AML remain largely unknown. Here we investigated its anticancer effects and underlying mechanisms on human AML cells in vitro and in vivo. The results showed that matrine inhibited cell viability and induced cell apoptosis in AML cell lines as well as primary AML cells from patients with AML in a dose- and time-dependent manner. Matrine induced apoptosis by collapsing the mitochondrial membrane potential, inducing cytochrome c release from mitochondria, reducing the ratio of Bcl-2/Bax, increasing activation of caspase-3, and decreasing the levels of p-Akt and p-ERK1/2. The apoptotic effects of matrine on AML cells were partially blocked by a caspase-3 inhibitor Z-DEVD-FMK and a PI3K/Akt activator IGF-1, respectively. Matrine potently inhibited in vivo tumor growth following subcutaneous inoculation of HL-60 cells in SCID mice. These findings indicate that matrine can inhibit cell proliferation and induce apoptosis of AML cells and may be a novel effective candidate as chemotherapeutic agent against AML.

  18. Cytotoxic T cell response against the chimeric ETV6-AML1 protein in childhood acute lymphoblastic leukemia.

    OpenAIRE

    Yotnda, P.; Garcia,F.; Peuchmaur, M.; Grandchamp, B.; Duval, M.; Lemonnier, F; Vilmer, E; Langlade-Demoyen, P

    1998-01-01

    Cytotoxic T lymphocytes (CTL) are potent effector cells that could provide long term antitumor immunity if induced by appropriate vaccines. CTL recognize 8-14 amino acid-long peptides processed intracellularly and presented by MHC class I molecules. A well-characterized example of a potential tumor antigen in childhood pre-B Acute Lymphoblastic Leukemia (ALL) results from the chromosomal translocation 12;21 leading to the fusion of the ETV6 and AML1 genes. This translocation is observed in > ...

  19. Atmospheric Measurements Laboratory (AML)

    Data.gov (United States)

    Federal Laboratory Consortium — The Atmospheric Measurements Laboratory (AML) is one of the nation's leading research facilities for understanding aerosols, clouds, and their interactions. The AML...

  20. Celastrol Induces Cell Apoptosis and Inhibits the Expression of the AML1-ETO/C-KIT Oncoprotein in t(8;21 Leukemia

    Directory of Open Access Journals (Sweden)

    Xianjun Yu

    2016-04-01

    Full Text Available Resistance to chemotherapy is a major challenge to improving overall survival in Acute Myeloid Leukemia (AML. Therefore, the development of innovative therapies and the identification of more novel agents for AML are urgently needed. Celastrol, a compound extracted from the Chinese herb Tripterygium wilfordii Hook, exerts anticancer activity. We investigated the effect of celastrol in the t(8;21 AML cell lines Kasumi-1 and SKNO-1. We demonstrated that inhibition of cell proliferation activated caspases and disrupted mitochondrial function. In addition, we found that celastrol downregulated the AML1-ETO fusion protein, therefore downregulating C-KIT kinases and inhibiting AKT, STAT3 and Erk1/2. These findings provide clear evidence that celastrol might provide clinical benefits to patients with t(8;21 leukemia.

  1. Immune responses to WT1 in patients with AML or MDS after chemotherapy and allogeneic stem cell transplantation.

    Science.gov (United States)

    Casalegno-Garduño, Rosaely; Schmitt, Anita; Spitschak, Alf; Greiner, Jochen; Wang, Lei; Hilgendorf, Inken; Hirt, Carsten; Ho, Anthony D; Freund, Mathias; Schmitt, Michael

    2016-04-01

    Wilms' tumor gene 1 (WT1) is overexpressed in leukemia and WT1-derived CD8(+) T-cell epitopes for immunotherapies targeting WT1 have been defined. Here, we analyzed expression of WT1 in 226 peripheral blood and bone marrow samples from patients with acute myeloid leukemia or myelodysplastic syndrome (AML/MDS) before and after allogeneic stem cell transplantation (SCT). Transcripts were assessed by quantitative polymerase chain reaction, and WT1-specific CD8+ cytotoxic T cells (CTL) were monitored by tetramer staining and enzyme-linked immunospot (ELISPOT) assays. Reduction of WT1 levels correlated with a longer survival (p CTL than in patients developing a relapse were detected. These cells were effector T cells secreting interferon gamma and granzyme B. In summary, WT1 is a suitable marker for the detection of minimal residual disease after SCT or chemotherapy. A rising WT1 signal correlated with a dismal prognosis of the patients. WT1-specific CD8(+) T cells might contribute to the maintenance of a cCR. Targeting WT-1 by peptide/protein vaccination as well as adoptive transfer of genetically modified T cells are future options in the individualized therapy for AML/MDS patients. © 2015 UICC.

  2. Pleiotropic effects of spongean alkaloids on mechanisms of cell death, cell cycle progression and DNA damage response (DDR) of acute myeloid leukemia (AML) cells.

    Science.gov (United States)

    Stuhldreier, Fabian; Kassel, Stefanie; Schumacher, Lena; Wesselborg, Sebastian; Proksch, Peter; Fritz, Gerhard

    2015-05-28

    We investigated cytotoxic mechanisms evoked by the spongean alkaloids aaptamine (Aa) and aeroplysinin-1 (Ap), applied alone and in combination with daunorubicin, employing acute myeloid leukemia (AML) cells. Aa and Ap reduced the viability of AML cells in a dose dependent manner with IC50 of 10-20 µM. Ap triggered apoptotic cell death more efficiently than Aa. Both alkaloids increased the protein level of S139-phosphorylated H2AX (γH2AX), which however was independent of the induction of DNA damage. Expression of the senescence markers p21 and p16 was increased, while the phosphorylation level of p-Chk-2 was reduced following Aa treatment. As a function of dose, Aa and Ap protected or sensitized AML cells against daunorubicin. Protection by Aa was paralleled by reduced formation of ROS and lower level of DNA damage. Both Aa and Ap attenuated daunorubicin-stimulated activation of the DNA damage response (DDR) as reflected on the levels of γH2AX, p-Kap-1 and p-Chk-1. Specifically Ap restored the decrease in S10 phosphorylation of histone H3 resulting from daunorubicin treatment. The cytoprotective effects of Aa and Ap were independent of daunorubicin import/export. Both Aa and Ap abrogated daunorubicin-induced accumulation of cells in S-phase. Inhibition of DNA synthesis was specific for Ap. The data show that Aa and Ap have both congruent and agent-specific pleiotropic effects that are preferential for anticancer drugs. Since Ap showed a broader spectrum of anticancer activities, this compound is suggested as novel lead compound for forthcoming in vivo studies elucidating the usefulness of spongean alkaloids in AML therapy.

  3. Mutation Analysis of hCDC4 in AML Cells Identifies a New Intronic Polymorphism

    Directory of Open Access Journals (Sweden)

    Daniel Nowak, Maximilian Mossner, Claudia D. Baldus, Olaf Hopfer, Eckhard Thiel, Wolf-Karsten Hofmann

    2006-01-01

    Full Text Available hCDC4 (FBW7, FBXW7 is a new potential tumor suppressor gene which provides substrate specificity for SCF (Skp–Cullin–F-box ubiquitin ligases and thereby regulates the degradation of potent oncogenes such as cyclin E, Myc, c-Jun and Notch. Mutations in the hCDC4 gene have been found in several solid tumors such as pancreas, colorectal or endometrial cancer. We carried out a mutation analysis of the hCDC4 gene in 35 samples of patients with Acute Myeloid Leukemia (AML to elucidate a possible role of hCDC4 mutations in this disease. By direct DNA sequencing and digestion with Surveyor nuclease one heterozygous mutation in the 5' untranslated region of exon 1, transcript variant 3 was detected. Additionally, we could identify a new intronic SNP downstream of exon 10. The new variation was present in 20% of AML samples and was furthermore confirmed in a panel of 51 healthy individuals where it displayed a frequency of 14%. In conclusion we provide first data that in contrast to several solid tumors, mutations in the hCDC4 gene may not play a pivotal role in the pathogenesis of AML. Furthermore, we describe a new intronic polymorphism with high frequency in the intron sequence of the hCDC4 gene.

  4. Double loading of dendritic cell MHC class I and MHC class II with an AML antigen repertoire enhances correlates of T-cell immunity in vitro via amplification of T-cell help.

    Science.gov (United States)

    Decker, William K; Xing, Dongxia; Li, Sufang; Robinson, Simon N; Yang, Hong; Yao, Xin; Segall, Harry; McMannis, John D; Komanduri, Krishna V; Champlin, Richard E; Shpall, Elizabeth J

    2006-04-12

    Therapeutic vaccination with dendritic cells presenting tumor-specific antigens is now recognized as an important investigational therapy for the treatment of neoplastic disease. Dendritic cell cross-presentation is credited with the ability of tumor lysate-loaded dendritic cells to prime both CD4 and CD8-specific T-lymphocyte responses, enabling the generation of cancer specific CTL activity without the loading of the classical MHC class I compartment. Recently, however, several reports have raised doubts as to the efficiency of cross-presentation as a mechanism for CTL priming in vivo. To examine this issue, we have doubly-loaded human dendritic cells with both AML-specific tumor lysate and AML-specific tumor mRNA. Our results show that these doubly-loaded dendritic cells can mediate superior primary, recall, and effector lytic responses in vitro in comparison to those of dendritic cells loaded with either tumor lysate or tumor mRNA alone. Enhanced recall responses appeared to be influenced by CD40/CD40L signaling, underscoring the importance of T-cell help in the generation and perpetuation of the adaptive immune response.

  5. Biology and relevance of human acute myeloid leukemia stem cells.

    Science.gov (United States)

    Thomas, Daniel; Majeti, Ravindra

    2017-03-23

    Evidence of human acute myeloid leukemia stem cells (AML LSCs) was first reported nearly 2 decades ago through the identification of rare subpopulations of engrafting cells in xenotransplantation assays. These AML LSCs were shown to reside at the apex of a cellular hierarchy that initiates and maintains the disease, exhibiting properties of self-renewal, cell cycle quiescence, and chemoresistance. This cancer stem cell model offers an explanation for chemotherapy resistance and disease relapse and implies that approaches to treatment must eradicate LSCs for cure. More recently, a number of studies have both refined and expanded our understanding of LSCs and intrapatient heterogeneity in AML using improved xenotransplant models, genome-scale analyses, and experimental manipulation of primary patient cells. Here, we review these studies with a focus on the immunophenotype, biological properties, epigenetics, genetics, and clinical associations of human AML LSCs and discuss critical questions that need to be addressed in future research. © 2017 by The American Society of Hematology.

  6. The Runx-PU.1 pathway preserves normal and AML/ETO9a leukemic stem cells.

    Science.gov (United States)

    Staber, Philipp B; Zhang, Pu; Ye, Min; Welner, Robert S; Levantini, Elena; Di Ruscio, Annalisa; Ebralidze, Alexander K; Bach, Christian; Zhang, Hong; Zhang, Junyan; Vanura, Katrina; Delwel, Ruud; Yang, Henry; Huang, Gang; Tenen, Daniel G

    2014-10-01

    Runx transcription factors contribute to hematopoiesis and are frequently implicated in hematologic malignancies. All three Runx isoforms are expressed at the earliest stages of hematopoiesis; however, their function in hematopoietic stem cells (HSCs) is not fully elucidated. Here, we show that Runx factors are essential in HSCs by driving the expression of the hematopoietic transcription factor PU.1. Mechanistically, by using a knockin mouse model in which all three Runx binding sites in the -14kb enhancer of PU.1 are disrupted, we observed failure to form chromosomal interactions between the PU.1 enhancer and its proximal promoter. Consequently, decreased PU.1 levels resulted in diminished long-term HSC function through HSC exhaustion, which could be rescued by reintroducing a PU.1 transgene. Similarly, in a mouse model of AML/ETO9a leukemia, disrupting the Runx binding sites resulted in decreased PU.1 levels. Leukemia onset was delayed, and limiting dilution transplantation experiments demonstrated functional loss of leukemia-initiating cells. This is surprising, because low PU.1 levels have been considered a hallmark of AML/ETO leukemia, as indicated in mouse models and as shown here in samples from leukemic patients. Our data demonstrate that Runx-dependent PU.1 chromatin interaction and transcription of PU.1 are essential for both normal and leukemia stem cells.

  7. Mesenchymal Stem Cells Support Survival and Proliferation of Primary Human Acute Myeloid Leukemia Cells through Heterogeneous Molecular Mechanisms

    Science.gov (United States)

    Brenner, Annette K.; Nepstad, Ina; Bruserud, Øystein

    2017-01-01

    Acute myeloid leukemia (AML) is a bone marrow malignancy, and various bone marrow stromal cells seem to support leukemogenesis, including osteoblasts and endothelial cells. We have investigated how normal bone marrow mesenchymal stem cells (MSCs) support the in vitro proliferation of primary human AML cells. Both MSCs and primary AML cells show constitutive release of several soluble mediators, and the mediator repertoires of the two cell types are partly overlapping. The two cell populations were cocultured on transwell plates, and MSC effects on AML cells mediated through the local cytokine/soluble mediator network could thus be evaluated. The presence of normal MSCs had an antiapoptotic and growth-enhancing effect on primary human AML cells when investigating a group of 51 unselected AML patients; this was associated with increased phosphorylation of mTOR and its downstream targets, and the effect was independent of cytogenetic or molecular-genetic abnormalities. The MSCs also supported the long-term proliferation of the AML cells. A subset of the patients also showed an altered cytokine network with supra-additive levels for several cytokines. The presence of cytokine-neutralizing antibodies or receptor inhibitors demonstrated that AML cells derived from different patients were heterogeneous with regard to effects of various cytokines on AML cell proliferation or regulation of apoptosis. We conclude that even though the effects of single cytokines derived from bone marrow MSCs on human AML cells differ among patients, the final cytokine-mediated effects of the MSCs during coculture is growth enhancement and inhibition of apoptosis.

  8. Peripheral Blood WT1 Expression Predicts Relapse in AML Patients Undergoing Allogeneic Stem Cell Transplantation

    Directory of Open Access Journals (Sweden)

    Michele Malagola

    2014-01-01

    Full Text Available To evaluate if WT1 expression may predict relapse after allo-SCT, we analyzed WT1 levels on peripheral blood (PB and bone marrow (BM before and after allo-SCT in 24 AML patients with WT1 overexpression at diagnosis. Five copies of WT1/ABL × 104 from PB were identified as the threshold value that correlated with relapse after allo-SCT. The same correlation was not identified when WT1 expression was assessed from bone marrow (BM. Eight out of 11 (73% patients with a pre-allo-SCT PB-WT1 ≥ 5 and 4/13 (31% patients with a pre-allo-SCT PB-WT1 < 5 relapsed, respectively (P = 0.04. The incidence of relapse was higher in patients with PB-WT1 ≥ 5 measured after allo-SCT, at the 3rd (56% versus 38%; P = 0.43 and at the 6th month (71% versus 20%; P = 0.03. Patients with pretransplant PB-WT1 < 5 had significantly better 2-year OS and LFS than patients with a PB-WT1 ≥ 5 (81% versus 0% and 63% versus 20% (P = 0.02. Our data suggest the usefulness of WT1 monitoring from PB to predict the relapse in allotransplanted AML patients and to modulate the intensity of conditioning and/or the posttransplant immunosuppression in an attempt to reduce the posttransplant relapse risk.

  9. Targeting Aberrant Glutathione Metabolism to Eradicate Human Acute Myelogenous Leukemia Cells*

    Science.gov (United States)

    Pei, Shanshan; Minhajuddin, Mohammad; Callahan, Kevin P.; Balys, Marlene; Ashton, John M.; Neering, Sarah J.; Lagadinou, Eleni D.; Corbett, Cheryl; Ye, Haobin; Liesveld, Jane L.; O'Dwyer, Kristen M.; Li, Zheng; Shi, Lei; Greninger, Patricia; Settleman, Jeffrey; Benes, Cyril; Hagen, Fred K.; Munger, Joshua; Crooks, Peter A.; Becker, Michael W.; Jordan, Craig T.

    2013-01-01

    The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34+) leukemic versus normal specimens. Our data indicate that CD34+ AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34+ AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34+ cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34+ AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34+ cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells. PMID:24089526

  10. Preclinical validation: LV/IL-12 transduction of patient leukemia cells for immunotherapy of AML

    Directory of Open Access Journals (Sweden)

    Ju Huang

    2016-01-01

    Full Text Available Interleukin-12 (IL-12 is a potent cytokine that may be harnessed to treat cancer. To date, nearly 100 IL-12-based clinical trials have been initiated worldwide. Yet systemic administration of IL-12 is toxic. Different strategies are being developed to reduce such toxicities by restricting IL-12 distribution. Our previous studies employed lentivector-mediated expression of murine IL-12 in tumor cells and demonstrated effective protection in both mouse leukemia and solid tumor challenge models. In this study, we carried out preclinical validation studies using a novel lentivector to engineer expression of human IL-12 in acute myeloid leukemia blast cells isolated from 21 patients. Acute myeloid leukemia cells were transduced with a bicistronic lentivector that encodes the human IL-12 cDNA as a fusion, as well as a LNGFR (ΔLNGFR/mutant thymidylate kinase cassette as a marking and cell-fate control element. A range of 20–70% functional transduction efficiencies was achieved. Transduced acute myeloid leukemia cells produced bioactive IL-12 protein and displayed dose-dependent sensitivity to the prodrug 3′-azido-3′-deoxythymidine. In vitro immortalization assays using transduced mouse hematopoietic stem cells demonstrated minimal genotoxic risk from our IL-12 vector. Scale-up transduction and cell processing was subsequently validated in a GMP facility to support our (now approved Clinical Trial Application (CTA.

  11. Identification of the Molecular Mechanisms Responsible for the Inhibition of Homing of AML Cells Triggered by CD44-Ligation

    KAUST Repository

    Al-Jifri, Ablah

    2011-08-03

    Acute Myeloid Leukemia (AML) is a cancerous disease that is defined by the inability to produce functional and mature blood cells, as well as the uncontrolled proliferation due to failure to undergo apoptosis of abnormal cells. The most common therapy for Leukemia, chemotherapy, has proven only to be partially efficient since it does not target the leukemic stem cells (LSCs) that have a high self-renewal and repopulation capacity and result in remission of the disease. Therefore targeting LSCs will provide more efficient therapy. One way to achieve this would be to inhibit their homing capability to the bone marrow. It has recently been shown that CD44, an adhesive molecule, plays a crucial role in cell trafficking and lodgement of both normal and leukemic stem cells. More importantly anti-CD44 monoclonal antibodies, along with its ability to induce differentiation of leukemic blasts, it inhibits specifically the homing capacity of LSCs to their micro-environmental niches. However, these molecular mechanisms that underlie the inhibition of homing have yet to be determined. To address these questions we conducted in vitro adhesion and blot-rolling assays to analyze the adherence and rolling capacity of these LSCs before and after treatment with anti-CD44 monoclonal antibody (mAb). Since glycosyltransferases play a crucial role in post translational carbohydrate decoration on adhesion molecules, we analyzed the expression (using quantitative PCR) of the different glycosyltransferases expressed in LSC\\'s before and after CD44 ligation (mAb treatment). Furthermore, we analyzed differentiation by flow cytometric analysis of treated and non-treated LSC\\'s. We anticipate that our results will set forth new insights into targeted therapies for AML.

  12. [Dexamethasone and vorinostat cooperatively promote differentiation and apoptosis in Kasumi-1 leukemia cells through ubiquitination and degradation of AML1-ETO].

    Science.gov (United States)

    Chen, Li-ping; Zhang, Jian-wei; Xu, Fa-mei; Xing, Hai-yan; Tian, Zheng; Wang, Min; Wang, Jian-xiang

    2013-09-01

    To probe the effects of dexamethasone (DEX) combined with histone deacetylase (HDAC) inhibitor vorinostat on inhibiting proliferation and inducing differentiation and apoptosis in Kasumi-1 leukemia cells, and its possible mechanisms in order to provide a theoretical basis for the treatment of AML1-ETO positive AML. The cell survival, differentiation and apoptosis rates were tested by MTT or flow cytometry analysis after Kasumi-1 cells were treated by DMSO, DEX (20 nmol/L), vorinostat (1 μmol/L) or DEX (20 nmol/L) in combination with vorinostat (1 μmol/L). WB and IP-WB were performed to detect AML1-ETO and its ubiquitination. Treatment with the combination of DEX and vorinostat for 48 h led to statistically significant differences of inhibited proliferation [(42.06±8.20)%], increased differentiation [(52.83±8.97)%] and apoptosis [(52.92±2.53)%] of Kasumi-1 cells when compared with vorinostat [(33.82±9.41)%, (43.93±9.04)% and (42.98±3.01)%, respectively], DEX [(17.30±3.49)%, (22.53±4.51)% and (19.57±2.17)%, respectively] or control [(6.96±0.39)%, (21.73±2.03)% and (6.96±0.39)%, respectively]. Also significant ubiquitination and decreased AML1-ETO protein in Kasumi-1 cells after the combination treatment over single agent or control were observed. The results indicated that DEX and vorinostat could synergistically inhibit the Kasumi-1 cells proliferation, induce Kasumi-1 cells differentiation and apoptosis through ubiquitination and degradation of AML1-ETO.

  13. Clinical grade expansion of CD45RA, CD45RO, and CD62L-positive T-cell lines from HLA-compatible donors: high cytotoxic potential against AML and ALL cells.

    Science.gov (United States)

    Barbui, Anna M; Borleri, Gianmaria; Conti, Elena; Ciocca, Alice; Salvi, Anna; Micò, Caterina; Introna, Martino; Rambaldi, Alessandro

    2006-04-01

    Identification of a clinical grade method for the ex vivo generation of donor-derived T cells cytotoxic against both myeloid and lymphoblastic cells still remains elusive. We investigated rapid generation and expansion of donor derived-allogeneic T-cell lines cytotoxic against patient leukemic cells. Acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) blasts were cultured 5 days in Stem Span, granulocyte macrophage colony-stimulating factor, interleukin-4, and calcium ionophore. All B-precursor ALL (N22) and AML (N13), but not T-cell ALL (N3), differentiated into mature leukemia-derived antigen-presenting cells (LD-APC). All but one LD-APC generated cytotoxic T lymphocyte (CTL) from adult human leukocyte antigen (HLA)-identical (N8) or unrelated donors (N2). Upon in vitro culture, donor-derived CTL acquired a memory T phenotype, showing concomitant high CD45RA, CD45RO, CD62L expression. CD8(+) cells, but not CD4(+) cells, were granzyme, perforine, and interferon-gamma-positive. Pooled CD4(+) and CD8(+) cells were cytotoxic against leukemic blasts (32%, 30:1 E:T ratio), but not against autologous or patient-derived phytohemagglutinin blasts. LD-APC from five ALL patients were used to generate CTL from cord blood. A mixed population of CD4(+) and CD8(+) cells was documented in 54% of wells. T cells acquired classical effector memory phenotype and showed a higher cytotoxicity against leukemia blasts (47%, 1:1 E:T ratio). Adult and cord blood CTL showed a skewing from a complete T-cell receptor repertoire to an oligo-clonal/clonal pattern. Availability of these cells should allow clinical trials for salvage treatment of leukemia patients relapsing after allogeneic stem cell transplantation.

  14. Small molecule inhibition of cAMP response element binding protein in human acute myeloid leukemia cells.

    Science.gov (United States)

    Mitton, B; Chae, H-D; Hsu, K; Dutta, R; Aldana-Masangkay, G; Ferrari, R; Davis, K; Tiu, B C; Kaul, A; Lacayo, N; Dahl, G; Xie, F; Li, B X; Breese, M R; Landaw, E M; Nolan, G; Pellegrini, M; Romanov, S; Xiao, X; Sakamoto, K M

    2016-12-01

    The transcription factor CREB (cAMP Response-Element Binding Protein) is overexpressed in the majority of acute myeloid leukemia (AML) patients, and this is associated with a worse prognosis. Previous work revealed that CREB overexpression augmented AML cell growth, while CREB knockdown disrupted key AML cell functions in vitro. In contrast, CREB knockdown had no effect on long-term hematopoietic stem cell activity in mouse transduction/transplantation assays. Together, these studies position CREB as a promising drug target for AML. To test this concept, a small molecule inhibitor of CREB, XX-650-23, was developed. This molecule blocks a critical interaction between CREB and its required co-activator CBP (CREB Binding Protein), leading to disruption of CREB-driven gene expression. Inhibition of CBP-CREB interaction induced apoptosis and cell-cycle arrest in AML cells, and prolonged survival in vivo in mice injected with human AML cells. XX-650-23 had little toxicity on normal human hematopoietic cells and tissues in mice. To understand the mechanism of XX-650-23, we performed RNA-seq, ChIP-seq and Cytometry Time of Flight with human AML cells. Our results demonstrate that small molecule inhibition of CBP-CREB interaction mostly affects apoptotic, cell-cycle and survival pathways, which may represent a novel approach for AML therapy.

  15. The chimeric genes AML1/DS1 and AML1/EAP inhibit AML1B activation at the CSF1R promoter, but only AML1/MDS1 has tumor-promoter properties

    Energy Technology Data Exchange (ETDEWEB)

    Zent, C.S.; Matheiu, C.; Rowley, J.D. [Univ. of Chicago, IL (United States)] [and others

    1996-02-06

    The (3;21)(q26;q22) translocation associated with treatment-related myelodysplastic syndrome, treatment-related acute myeloid leukemia, and blast crisis of chronic myeloid leukemia results in the expression of the chimeric genes AML1/EAP, AML1MDS1, and AML1/EVI1. AML1 (CBFA2), which codes for the {alpha} subunit of the heterodimeric transcription factor CBF, is also involved in the t(8;21), and the gene coding for the {beta} subunit (CBFB) is involved in the inv(16). These are two of the most common recurring chromosomal rearrangements in acute myeloid leukemia. CBF corresponds to the murine Pebp2 factor, and CBF binding sites are found in a number of eukaryotic and viral enhancers and promoters. We studied the effects of AML1/EAP and AML1/MDS1 at the AML1 binding site of the CSF1R (macrophage-colony-stimulating factor receptor gene) promoter by using reporter gene assays, and we analyzed the consequences of the expression of both chimeric proteins in an embryonic rat fibroblast cell line (Rat1A) in culture and after injection into athymic nude mice. Unlike AML1, which is an activator of the CSF1R promoter, the chimeric proteins did not transactivate the CSF1R promoter site but acted as inhibitors of AMLI (CBFA2). AML1/EAP and AML1/MDS1 expressed in adherent Rat1A cells decreased contact inhibition of growth, and expression of AML1/MDS1 was associated with acquisition of the ability to grow in suspension culture. Expression of AML1/MDS1 increased the tumorigenicity of Rat1A cells injected into athymic nude mice, whereas AML1/EAP expression provented tumor growth. These results suggest that expression of AML1/MDS1 can interfere with normal AML1 function, and that AML1/MDS1 has tumor-promoting properties in an embryonic rat fibroblast cell line. 26 refs., 5 figs.

  16. Cyclin-dependent kinase phosphorylation of RUNX1/AML1 on 3 sites increases transactivation potency and stimulates cell proliferation

    Science.gov (United States)

    Zhang, Linsheng; Fried, Florence B.; Guo, Hong

    2008-01-01

    RUNX1/AML1 regulates lineage-specific genes during hematopoiesis and stimulates G1 cell-cycle progression. Within RUNX1, S48, S303, and S424 fit the cyclin-dependent kinase (cdk) phosphorylation consensus, (S/T)PX(R/K). Phosphorylation of RUNX1 by cdks on serine 303 was shown to mediate destabilization of RUNX1 in G2/M. We now use an in vitro kinase assay, phosphopeptide-specific antiserum, and the cdk inhibitor roscovitine to demonstrate that S48 and S424 are also phosphorylated by cdk1 or cdk6 in hematopoietic cells. S48 phosphorylation of RUNX1 paralleled total RUNX1 levels during cell-cycle progression, S303 was more effectively phosphorylated in G2/M, and S424 in G1. Single, double, and triple mutation of the cdk sites to the partially phosphomimetic aspartic acid mildly reduced DNA affinity while progressively increasing transactivation of a model reporter. Mutation to alanine increased DNA affinity, suggesting that in other gene or cellular contexts phosphorylation of RUNX1 by cdks may reduce transactivation. The tripleD RUNX1 mutant rescued Ba/F3 cells from inhibition of proliferation by CBFβ-SMMHC more effectively than the tripleA mutant. Together these findings indicate that cdk phosphorylation of RUNX1 potentially couples stem/progenitor proliferation and lineage progression. PMID:18003885

  17. In vitro characterization of human AML-reactive CTL clones generated from the naive subset of healthy donors and adoptive transfer into leukemia-engrafted NSG mice

    OpenAIRE

    Albrecht, Jana

    2012-01-01

    Acute myeloid leukemia (AML) is a very aggressive cancer of the hematopoietic system. Chemotherapy and immunotherapeutical approaches including hematopoietic stem cell transplantation (HSCT) and donor lymphocyte infusion (DLI) are the only curative options available. The beneficial graft-versus-leukemia (GVL) effect of cellular immunotherapy is mostly mediated by donor-derived CD8+ T lymphocytes that recognize minor histocompatibility antigens (mHags) and leukemia-associated antigens (LAAs) p...

  18. Decitabine enhances anti-CD33 monoclonal antibody BI 836858-mediated natural killer ADCC against AML blasts.

    Science.gov (United States)

    Vasu, Sumithira; He, Shun; Cheney, Carolyn; Gopalakrishnan, Bhavani; Mani, Rajeswaran; Lozanski, Gerard; Mo, Xiaokui; Groh, Veronica; Whitman, Susan P; Konopitzky, Renate; Kössl, Christian; Bucci, Donna; Lucas, David M; Yu, Jianhua; Caligiuri, Michael A; Blum, William; Adam, Paul J; Borges, Eric; Rueter, Bjoern; Heider, Karl-Heinz; Marcucci, Guido; Muthusamy, Natarajan

    2016-06-09

    Acute myeloid leukemia (AML) is the most common type of acute leukemia, affecting older individuals at a median age of 67 years. Resistance to intensive induction chemotherapy is the major cause of death in elderly AML; hence, novel treatment strategies are warranted. CD33-directed antibody-drug conjugates (gemtuzumab ozogamicin) have been shown to improve overall survival, validating CD33 as a target for antibody-based therapy of AML. Here, we report the in vitro efficacy of BI 836858, a fully human, Fc-engineered, anti-CD33 antibody using AML cell lines and primary AML blasts as targets. BI 836858-opsonized AML cells significantly induced both autologous and allogeneic natural killer (NK)-cell degranulation and NK-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). In vitro treatment of AML blasts with decitabine (DAC) or 5-azacytidine, 2 hypomethylating agents that show efficacy in older patients, did not compromise BI 836858-induced NK-cell-mediated ADCC. Evaluation of BI 836858-mediated ADCC in serial marrow AML aspirates in patients who received a 10-day course of DAC (pre-DAC, days 4, 11, and 28 post-DAC) revealed significantly higher ADCC in samples at day 28 post-DAC when compared with pre-DAC treatment. Analysis of ligands to activating receptors (NKG2D) showed significantly increased NKG2D ligand [NKG2DL] expression in day 28 post-DAC samples compared with pre-DAC samples; when NKG2DL receptor was blocked using antibodies, BI 836858-mediated ADCC was significantly decreased, suggesting that DAC enhances AML blast susceptibility to BI 836858 by upregulating NKG2DL. These data provide a rationale for combination therapy of Fc-engineered antibodies such as BI 836858 with azanucleosides in elderly patients with AML.

  19. Growth response of acute myeloblastic leukemia cells to recombinant human thrombopoietin.

    Science.gov (United States)

    Matsumura, I; Kanakura, Y; Kato, T; Ikeda, H; Ishikawa, J; Horikawa, Y; Hashimoto, K; Moriyama, Y; Tsujimura, T; Nishiura, T

    1995-07-15

    Thrombopoietin (TPO) is a newly identified hematopoietic growth factor that stimulates both megakaryopoiesis and thrombopoiesis through its interaction with a specific cell surface receptor encoded by the c-mpl proto-oncogene. In an effort to investigate the effect of TPO on human myeloid leukemia cells, the expression of c-mpl and the proliferative response to recombinant human (rh) TPO were investigated in a series of patients with acute myeloblastic leukemia (AML). Of 50 cases of AML, the c-mpl mRNA was detectable by means of Northern blot analysis in 26 cases, and the in vitro treatment with rhTPO led to proliferation of AML cells in 22 cases. The c-mpl expression and proliferative response to rhTPO was observed in all subtypes of AML and did not correlate with French-American-British classification, whereas all cases of M7-type AML cells expressed c-mpl and proliferated in response to rhTPO. Furthermore, rhTPO-induced proliferation of AML cells was augmented with the addition of interleukin-3 (IL-3), IL-6, stem cell factor, or granulocyte-macrophage colony-stimulating factor. These results suggested that c-mpl may be functional in terms of supporting proliferation of various types of AML cells and that TPO may contribute, at least in part, to abnormal growth of the cells, especially in combination with other hematopoietic growth factors.

  20. Donor haplotype B of NK KIR receptor reduces the relapse risk in HLA-identical sibling hematopoietic stem cell transplantation of AML patients.

    Directory of Open Access Journals (Sweden)

    Ulla eImpola

    2014-08-01

    Full Text Available Successful allogeneic hematological stem cell transplantation (HSCT depends not only on good HLA match but also on T-cell mediated graft-versus-leukemia (GvL effect. Natural killer (NK cells are able to kill malignant cells by receiving activation signal from the killer-cell immunoglobulin-like receptors (KIR recognizing HLA molecules on a cancer cell. It has been recently reported that the risk of relapse in allogeneic hematopoietic stem cell transplantation (HSCT is reduced in acute myeloid leukemia (AML patients whose donors have several activating KIR genes or KIR B-motifs in unrelated donor (URD setting, obviously due to enhanced graft-versus-leukemia effect by NK cells. We studied the effect on relapse rate of donor KIR haplotypes in the HLA identical adult sibling HSCT, done in a single center, in Helsinki University Central Hospital, Helsinki, Finland. Altogether 134 patients with 6 different diagnoses were identified. Their donors were KIR genotyped using the Luminex and the SSP techniques. The clinical endpoint, that is, occurrence of relapse, was compared with the presence or absence of single KIR genes. Also, time from transplantation to relapse was analyzed. The patients with AML whose donors have KIR2DL2 or KIR2DS2 had statistically significantly longer relapse-free survival (P=0.015. Our data support previous reports that donors with KIR B-haplotype defining genes have a lower occurrence of relapse in HSCT of AML patients. Determination of donor KIR haplotypes could be a useful addition for a risk assessment of HSCT especially in AML patients.

  1. Donor Haplotype B of NK KIR Receptor Reduces the Relapse Risk in HLA-Identical Sibling Hematopoietic Stem Cell Transplantation of AML Patients.

    Science.gov (United States)

    Impola, Ulla; Turpeinen, Hannu; Alakulppi, Noora; Linjama, Tiina; Volin, Liisa; Niittyvuopio, Riitta; Partanen, Jukka; Koskela, Satu

    2014-01-01

    Successful allogeneic hematopoietic stem cell transplantation (HSCT) depends not only on good HLA match but also on T-cell mediated graft-versus-leukemia (GvL) effect. Natural killer (NK) cells are able to kill malignant cells by receiving activation signal from the killer-cell immunoglobulin-like receptors (KIR) recognizing HLA molecules on a cancer cell. It has been recently reported that the risk of relapse in allogeneic hematopoietic stem cell transplantation (HSCT) is reduced in acute myeloid leukemia (AML) patients whose donors have several activating KIR genes or KIR B-motifs in unrelated donor setting, obviously due to enhanced GvL effect by NK cells. We studied the effect on relapse rate of donor KIR haplotypes in the HLA-identical adult sibling HSCT, done in a single center, in Helsinki University Central Hospital, Helsinki, Finland. Altogether, 134 patients with 6 different diagnoses were identified. Their donors were KIR genotyped using the Luminex and the SSP techniques. The clinical endpoint, that is, occurrence of relapse, was compared with the presence or absence of single KIR genes. Also, time from transplantation to relapse was analyzed. The patients with AML whose donors have KIR2DL2 or KIR2DS2 had statistically significantly longer relapse-free survival (P = 0.015). Our data support previous reports that donors with KIR B-haplotype defining genes have a lower occurrence of relapse in HSCT of AML patients. Determination of donor KIR haplotypes could be a useful addition for a risk assessment of HSCT especially in AML patients.

  2. The biologic properties of recombinant human thrombopoietin in the proliferation and megakaryocytic differentiation of acute myeloblastic leukemia cells.

    Science.gov (United States)

    Matsumura, I; Kanakura, Y; Kato, T; Ikeda, H; Horikawa, Y; Ishikawa, J; Kitayama, H; Nishiura, T; Tomiyama, Y; Miyazaki, H; Matsuzawa, Y

    1996-10-15

    Thrombopoietin (TPO) is implicated as a primary regulator of megakaryopoiesis and thrombopoiesis. However, the biologic effects of TPO on human acute myeloblastic leukemia (AML) cells are largely unknown. To determine if recombinant human (rh) TPO has proliferation-supporting and differentiation-inducing activities in AML cells, 15 cases of AML cells that were exclusively composed of undifferentiated leukemia cells and showed growth response to rhTPO in a short-term culture (72 hours) were subjected to long-term suspension culture with or without rhTPO. Of 15 cases, rhTPO supported proliferation of AML cells for 2 to 4 weeks in 4 cases whose French-American-British subtypes were M0, M2, M4, and M7, respectively. In addition to the proliferation-supporting activity, rhTPO was found to induce AML cells to progress to some degree of megakaryocytic differentiation at both morphologic and surface-phenotypic level in 2 AML cases with M0 and M7 subtypes. The treatment of AML cells with rhTPO resulted in rapid tyrosine phosphorylation of the TPO-receptor, c-mpl, and STAT3 in all of cases tested. By contrast, the expression of erythroid/megakaryocyte-specific transcription factors (GATA-1, GATA-2, and NF-E2) was markedly induced or enhanced in only 2 AML cases that showed megakaryocytic differentiation in response to rhTPO. These results suggested that, at least in a fraction of AML cases, TPO could not only support the proliferation of AML cells irrespective of AML subtypes, but could also induce megakaryocytic differentiation, possibly through activation of GATA-1, GATA-2, and NF-E2.

  3. Sulindac sulfide reverses aberrant self-renewal of progenitor cells induced by the AML-associated fusion proteins PML/RARα and PLZF/RARα.

    Science.gov (United States)

    Steinert, Gunnar; Oancea, Claudia; Roos, Jessica; Hagemeyer, Heike; Maier, Thorsten; Ruthardt, Martin; Puccetti, Elena

    2011-01-01

    Chromosomal translocations can lead to the formation of chimeric genes encoding fusion proteins such as PML/RARα, PLZF/RARα, and AML-1/ETO, which are able to induce and maintain acute myeloid leukemia (AML). One key mechanism in leukemogenesis is increased self renewal of leukemic stem cells via aberrant activation of the Wnt signaling pathway. Either X-RAR, PML/RARα and PLZF/RARα or AML-1/ETO activate Wnt signaling by upregulating γ-catenin and β-catenin. In a prospective study, a lower risk of leukemia was observed with aspirin use, which is consistent with numerous studies reporting an inverse association of aspirin with other cancers. Furthermore, a reduction in leukemia risk was associated with use of non-steroidal anti-inflammatory drug (NSAID), where the effects on AML risk was FAB subtype-specific. To better investigate whether NSAID treatment is effective, we used Sulindac Sulfide in X-RARα-positive progenitor cell models. Sulindac Sulfide (SSi) is a derivative of Sulindac, a NSAID known to inactivate Wnt signaling. We found that SSi downregulated both β-catenin and γ-catenin in X-RARα-expressing cells and reversed the leukemic phenotype by reducing stem cell capacity and increasing differentiation potential in X-RARα-positive HSCs. The data presented herein show that SSi inhibits the leukemic cell growth as well as hematopoietic progenitors cells (HPCs) expressing PML/RARα, and it indicates that Sulindac is a valid molecular therapeutic approach that should be further validated using in vivo leukemia models and in clinical settings.

  4. Sulindac sulfide reverses aberrant self-renewal of progenitor cells induced by the AML-associated fusion proteins PML/RARα and PLZF/RARα.

    Directory of Open Access Journals (Sweden)

    Gunnar Steinert

    Full Text Available Chromosomal translocations can lead to the formation of chimeric genes encoding fusion proteins such as PML/RARα, PLZF/RARα, and AML-1/ETO, which are able to induce and maintain acute myeloid leukemia (AML. One key mechanism in leukemogenesis is increased self renewal of leukemic stem cells via aberrant activation of the Wnt signaling pathway. Either X-RAR, PML/RARα and PLZF/RARα or AML-1/ETO activate Wnt signaling by upregulating γ-catenin and β-catenin. In a prospective study, a lower risk of leukemia was observed with aspirin use, which is consistent with numerous studies reporting an inverse association of aspirin with other cancers. Furthermore, a reduction in leukemia risk was associated with use of non-steroidal anti-inflammatory drug (NSAID, where the effects on AML risk was FAB subtype-specific. To better investigate whether NSAID treatment is effective, we used Sulindac Sulfide in X-RARα-positive progenitor cell models. Sulindac Sulfide (SSi is a derivative of Sulindac, a NSAID known to inactivate Wnt signaling. We found that SSi downregulated both β-catenin and γ-catenin in X-RARα-expressing cells and reversed the leukemic phenotype by reducing stem cell capacity and increasing differentiation potential in X-RARα-positive HSCs. The data presented herein show that SSi inhibits the leukemic cell growth as well as hematopoietic progenitors cells (HPCs expressing PML/RARα, and it indicates that Sulindac is a valid molecular therapeutic approach that should be further validated using in vivo leukemia models and in clinical settings.

  5. Myeloablative radioimmunotherapies in the conditioning of patients with AML, MDS and multiple myeloma prior to stem cell transplantation; Myeloablative Radioimmuntherapien zur Konditionierung bei Patienten mit AML, MDS und multiplem Myelom vor Stammzelltransplantation

    Energy Technology Data Exchange (ETDEWEB)

    Buchmann, I. [Abt. fuer Nuklearmedizin, Universitaetsklinik Heidelberg (Germany)

    2008-06-15

    Aggressive consolidation chemotherapy and hematopoietic stem cell transplantation have improved the prognosis of patients with acute myeloid leukemia (AML), myelodyplastic syndrome (MDS) and multiple myeloma. Nevertheless, only a minor fraction of patients achieve long-term disease-free survival after stem cell transplantation with disease recurrence being the most common cause of treatment failure. In addition, therapy-related effects such as toxicity of chemotherapy and complications of stem cell transplantation increase mortality rates significantly. Myeloablative radioimmunotherapy uses radiolabeled monoclonal antibodies (mAb) with affinity for the hematopoietic marrow. It applies high radiation doses in the bone marrow but spares normal organs. Adding myeloablative radioimmunotherapy to the conditioning schemes of AML, MDS and multiple myeloma before stem cell transplantation allows for the achievement of a pronounced antileukemic/antimyeloma effect for the reduction of relapse rates without significant increase of acute organ toxicity and therapy-related mortality. In order to optimise therapy, a rational design of the nuclide-antibody combination is necessary. {sup 90}Y, {sup 188}Re and {sup 131}I are the most frequently used {beta}{sup -}-particles. Of these, {sup 90}Y is the most qualified nuclide for myeloablation. Backbone stabilised DTPA are ideal chelators to stably conjugate {sup 90}Y to antibodies so far. For myeloablative conditioning, anti-CD66-, -45- and -33-mAb are used. The anti-CD66-antibody BW250/183 binds to normal hematopoietic cells but not to leukemic blasts and myeloma cells. The {sup 90}Y-2B3M-DTPA-BW250/183 is the most suited radioimmunoconjugate for patients with an infiltration grade of leukemic blasts in the bone marrow < 25%. The specific doses (Gy/GBq) are 10.2 {+-} 1.8 (bone marrow), 2.7 {+-} 2 (liver) and < 1 (kidneys). In contrast, radiolabeled anti-CD33- and anti-CD45-antibodies bind to both, most of white blood cells and

  6. Outcome of children with high-risk acute myeloid leukemia given autologous or allogeneic hematopoietic cell transplantation in the aieop AML-2002/01 study.

    Science.gov (United States)

    Locatelli, F; Masetti, R; Rondelli, R; Zecca, M; Fagioli, F; Rovelli, A; Messina, C; Lanino, E; Bertaina, A; Favre, C; Giorgiani, G; Ripaldi, M; Ziino, O; Palumbo, G; Pillon, M; Pession, A; Rutella, S; Prete, A

    2015-02-01

    We analyzed the outcome of 243 children with high-risk (HR) AML in first CR1 enrolled in the AIEOP-2002/01 protocol, who were given either allogeneic (ALLO; n=141) or autologous (AUTO; n=102) hematopoietic SCT (HSCT), depending on the availability of a HLA-compatible sibling. Infants, patients with AML-M7, or complex karyotype or those with FLT3-ITD, were eligible to be transplanted also from alternative donors. All patients received a myeloablative regimen combining busulfan, cyclophosphamide and melphalan; [corrected] AUTO-HSCT patients received BM cells in most cases, while in children given ALLO-HSCT stem cell source was BM in 96, peripheral blood in 19 and cord blood in 26. With a median follow-up of 57 months (range 12-130), the probability of disease-free survival (DFS) was 73% and 63% in patients given either ALLO- or AUTO-HSCT, respectively (P=NS). Although the cumulative incidence (CI) of relapse was lower in ALLO- than in AUTO-HSCT recipients (17% vs 28%, respectively; P=0.043), the CI of TRM was 7% in both groups. Patients transplanted with unrelated donor cord blood had a remarkable 92.3% 8-year DFS probability. Altogether, these data confirm that HSCT is a suitable option for preventing leukemia recurrence in HR children with CR1 AML.

  7. Numerical impairment of nestin(+) bone marrow niches in acute GvHD after allogeneic hematopoietic stem cell transplantation for AML.

    Science.gov (United States)

    Medinger, M; Krenger, W; Jakab, A; Halter, J; Buser, A; Bucher, C; Passweg, J; Tzankov, A

    2015-11-01

    The nestin(+) perivascular bone marrow (BM) stem cell niche (N(+)SCN) may be involved in GvHD. To investigate whether acute GvHD (aGvHD) reduces the number of N(+)SCN, we examined patients with AML who had undergone allogeneic hematopoietic stem cell transplantation. In the test cohort (n=8), the number of N(+)SCN per mm(2) in BM biopsies was significantly reduced in aGvHD patients at the time of aGvHD compared with patients who did not have aGvHD (1.2±0.78 versus 2.6±0.93, P=0.04). In the validation cohort (n=40), the number of N(+)SCN was reduced (1.9±0.99 versus 2.6±0.90 N(+)SCN/mm(2), P=0.05) in aGvHD patients. Receiver operating curves suggested that the cutoff score that best discriminated between patients with and without aGvHD was 2.29 N(+)SCN/mm(2). Applying this cutoff score, 9/11 patients with clinically relevant aGvHD (⩾grade 2) and 13/20 with any type of GvHD had decreased N(+)SCN numbers compared with only 10/29 patients without clinically relevant aGvHD (P=0.007) and 6/20 patients without any type of GvHD (P=0.028). In patients tracked over time, N(+)SCN density returned to normal after aGvHD resolved or remained stable in patients who did not have aGvHD. Our results show a decrease in the number of N(+)SCN in aGvHD.

  8. Identification of small molecules that support human leukemia stem cell activity ex vivo.

    Science.gov (United States)

    Pabst, Caroline; Krosl, Jana; Fares, Iman; Boucher, Geneviève; Ruel, Réjean; Marinier, Anne; Lemieux, Sébastien; Hébert, Josée; Sauvageau, Guy

    2014-04-01

    Leukemic stem cells (LSCs) are considered a major cause of relapse in acute myeloid leukemia (AML). Defining pathways that control LSC self-renewal is crucial for a better understanding of underlying mechanisms and for the development of targeted therapies. However, currently available culture conditions do not prevent spontaneous differentiation of LSCs, which greatly limits the feasibility of cell-based assays. To overcome these constraints we conducted a high-throughput chemical screen and identified small molecules that inhibit differentiation and support LSC activity in vitro. Similar to reports with cord blood stem cells, several of these compounds suppressed the aryl-hydrocarbon receptor (AhR) pathway, which we show to be inactive in vivo and rapidly activated ex vivo in AML cells. We also identified a compound, UM729, that collaborates with AhR suppressors in preventing AML cell differentiation. Together, these findings provide newly defined culture conditions for improved ex vivo culture of primary human AML cells.

  9. Access/AML -

    Data.gov (United States)

    Department of Transportation — The AccessAML is a web-based internet single application designed to reduce the vulnerability associated with several accounts assinged to a single users. This is a...

  10. Combination of sapacitabine and HDAC inhibitors stimulates cell death in AML and other tumour types.

    Science.gov (United States)

    Green, S R; Choudhary, A K; Fleming, I N

    2010-10-26

    Alternative treatments are needed for elderly patients with acute myeloid leukaemia, as the disease prognosis is poor and the current treatment is unsuitable for many patients. In this study, we investigated whether combining the nucleoside analogue sapacitabine with histone deacetylase (HDAC) inhibitors could be an effective treatment. Synergy and mode-of-action analysis were studied in cultured cell lines and the efficacy of the combination was confirmed in a xenograft model. CNDAC (1-(2-C-cyano-2-deoxy-β-D-arabino-pentofuranosyl)-cytosine), the active component of sapacitabine, synergised with vorinostat in cell lines derived from a range of tumour types. Synergy was not dependent on a specific sequence of drug administration and was also observed when CNDAC was combined with an alternative HDAC inhibitor, valproate. Flow cytometry and western blot analysis confirmed that the combination induced a significant increase in apoptosis. Mode-of-action analysis detected changes in Bcl-xl, Mcl-1, Noxa, Bid and Bim, which are all regulators of the apoptotic process. The sapacitabine/vorinostat combination demonstrated significant benefit compared with the single-agent treatments in an MV4-11 xenograft, in the absence of any observed toxicity. Sapacitabine and HDAC inhibitors are an effective drug combination that is worthy of clinical exploration.

  11. Effects of recombinant human GM-CSF on proliferation of clonogenic cells in acute myeloblastic leukemia.

    Science.gov (United States)

    Griffin, J D; Young, D; Herrmann, F; Wiper, D; Wagner, K; Sabbath, K D

    1986-05-01

    Proliferation of acute myeloblastic leukemia (AML) cells in vitro is limited in most cases to a small subset of blasts that have several properties of stem cells. These leukemic colony-forming cells (AML-CFU) generally require addition of exogenous growth factors for proliferation in agar or methylcellulose. These factors can be supplied by media conditioned by phytohemagglutinin-stimulated normal leukocytes or by CSF-secreting tumor cell lines. However, the exact factor or factors required for stimulation of AML-CFU growth have not been defined. We compared the AML-CFU stimulatory activity of a human recombinant GM-CSF with that of GCT-CM, Mo-CM, and the PHA-leukocyte feeder system in 15 cases of AML. In each of the 12 cases that required exogenous growth factors for maximum AML-CFU growth, recombinant GM-CSF could replace either GM-CSF or Mo-CM, and could partially replace the PHA-leukocyte feeder system. These results indicate that this GM-CSF is a growth promoter of AML-CFU in these culture systems.

  12. Erlotinib exhibits antineoplastic off-target effects in AML and MDS: a preclinical study.

    Science.gov (United States)

    Boehrer, Simone; Adès, Lionel; Braun, Thorsten; Galluzzi, Lorenzo; Grosjean, Jennifer; Fabre, Claire; Le Roux, Génèviève; Gardin, Claude; Martin, Antoine; de Botton, Stéphane; Fenaux, Pierre; Kroemer, Guido

    2008-02-15

    Erlotinib, an inhibitor of the epidermal growth factor receptor (EGFR), induces differentiation, cell-cycle arrest, and apoptosis of EGFR-negative myeloblasts of patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), as well as in EGFR-negative cell lines representing these diseases (P39, KG-1, and HL 60). This off-target effect can be explained by inhibitory effects on JAK2. Apoptosis induction coupled to mitochondrial membrane permeabilization occurred independently from phenotypic differentiation. In apoptosis-sensitive AML cells, erlotinib caused a rapid (within less than 1 hour) nucleocytoplasmic translocation of nucleophosmin-1 (NPM-1) and p14(ARF). Apoptosis-insensitive myeloblasts failed to manifest this translocation yet became sensitive to apoptosis induction by erlotinib when NPM-1 was depleted by RNA interference. Moreover, erlotinib reduced the growth of xenografted human AML cells in vivo. Erlotinib also killed CD34(+) bone marrow blasts from MDS and AML patients while sparing normal CD34(+) progenitors. This ex vivo therapeutic effect was once more associated with the nucleocytoplasmic translocation of NPM-1 and p14(ARF). One patient afflicted with both MDS and non-small cell lung cancer manifested hematologic improvement in response to erlotinib. In summary, we here provide novel evidence in vitro, ex vivo, and in vivo for the potential therapeutic efficacy of erlotinib in the treatment of high-risk MDS and AML.

  13. Chemogenomic landscape of RUNX1-mutated AML reveals importance of RUNX1 allele dosage in genetics and glucocorticoid sensitivity.

    Science.gov (United States)

    Simon, Laura; Lavallée, Vincent-Philippe; Bordeleau, Marie-Eve; Krosl, Jana; Baccelli, Irene; Boucher, Geneviève; Lehnertz, Bernhard; Chagraoui, Jalila; MacRae, Tara; Ruel, Réjean; Chantigny, Yves A; Lemieux, Sébastien; Marinier, Anne; Hébert, Josée; Sauvageau, Guy

    2017-08-30

    RUNX1-mutated (RUNX1mut) Acute Myeloid Leukemia (AML) is associated with adverse outcome, highlighting the urgent need for a better genetic characterization of this AML subgroup and for the design of efficient therapeutic strategies for this disease. Towards this goal, we further dissected the mutational spectrum and gene expression profile of RUNX1mut AML and correlated these results to drug sensitivity to identify novel compounds targeting this AML subgroup. RNA-sequencing of 47 RUNX1mut primary AML specimens was performed and sequencing results were compared to those of RUNX1 wild-type samples. Chemical screens were also conducted using RUNX1mut specimens to identify compounds selectively affecting the viability of RUNX1mut AML. We show that samples with no remaining RUNX1 wild-type allele are clinically and genetically distinct and display a more homogeneous gene expression profile. Chemical screening revealed that most RUNX1mut specimens are sensitive to glucocorticoids (GCs) and we confirmed that GCs inhibit AML cell proliferation through their interaction with the Glucocorticoid Receptor (GR). We observed that specimens harboring RUNX1 mutations expected to result in low residual RUNX1 activity are most sensitive to GCs, and that co-associating mutations as well as that GR levels contribute to GC sensitivity. Accordingly, acquired glucocorticoid sensitivity was achieved by negatively regulating RUNX1 expression in human AML cells. Our findings show the profound impact of RUNX1 allele dosage on gene expression profile and glucocorticoid sensitivity in AML, thereby opening opportunities for preclinical testing which may lead to drug repurposing and improved disease characterization. Copyright ©2017, American Association for Cancer Research.

  14. Hif-1α and Hif-2α synergize to suppress AML development but are dispensable for disease maintenance.

    Science.gov (United States)

    Vukovic, Milica; Guitart, Amelie V; Sepulveda, Catarina; Villacreces, Arnaud; O'Duibhir, Eoghan; Panagopoulou, Theano I; Ivens, Alasdair; Menendez-Gonzalez, Juan; Iglesias, Juan Manuel; Allen, Lewis; Glykofrydis, Fokion; Subramani, Chithra; Armesilla-Diaz, Alejandro; Post, Annemarie E M; Schaak, Katrin; Gezer, Deniz; So, Chi Wai Eric; Holyoake, Tessa L; Wood, Andrew; O'Carroll, Dónal; Ratcliffe, Peter J; Kranc, Kamil R

    2015-12-14

    Leukemogenesis occurs under hypoxic conditions within the bone marrow (BM). Knockdown of key mediators of cellular responses to hypoxia with shRNA, namely hypoxia-inducible factor-1α (HIF-1α) or HIF-2α, in human acute myeloid leukemia (AML) samples results in their apoptosis and inability to engraft, implicating HIF-1α or HIF-2α as therapeutic targets. However, genetic deletion of Hif-1α has no effect on mouse AML maintenance and may accelerate disease development. Here, we report the impact of conditional genetic deletion of Hif-2α or both Hif-1α and Hif-2α at different stages of leukemogenesis in mice. Deletion of Hif-2α accelerates development of leukemic stem cells (LSCs) and shortens AML latency initiated by Mll-AF9 and its downstream effectors Meis1 and Hoxa9. Notably, the accelerated initiation of AML caused by Hif-2α deletion is further potentiated by Hif-1α codeletion. However, established LSCs lacking Hif-2α or both Hif-1α and Hif-2α propagate AML with the same latency as wild-type LSCs. Furthermore, pharmacological inhibition of the HIF pathway or HIF-2α knockout using the lentiviral CRISPR-Cas9 system in human established leukemic cells with MLL-AF9 translocation have no impact on their functions. We therefore conclude that although Hif-1α and Hif-2α synergize to suppress the development of AML, they are not required for LSC maintenance.

  15. Hif-1α and Hif-2α synergize to suppress AML development but are dispensable for disease maintenance

    Science.gov (United States)

    Vukovic, Milica; Guitart, Amelie V.; Sepulveda, Catarina; Villacreces, Arnaud; O'Duibhir, Eoghan; Panagopoulou, Theano I.; Ivens, Alasdair; Menendez-Gonzalez, Juan; Iglesias, Juan Manuel; Allen, Lewis; Glykofrydis, Fokion; Subramani, Chithra; Armesilla-Diaz, Alejandro; Post, Annemarie E.M.; Schaak, Katrin; Gezer, Deniz; So, Chi Wai Eric; Holyoake, Tessa L.; Wood, Andrew; O'Carroll, Dónal; Ratcliffe, Peter J.

    2015-01-01

    Leukemogenesis occurs under hypoxic conditions within the bone marrow (BM). Knockdown of key mediators of cellular responses to hypoxia with shRNA, namely hypoxia-inducible factor-1α (HIF-1α) or HIF-2α, in human acute myeloid leukemia (AML) samples results in their apoptosis and inability to engraft, implicating HIF-1α or HIF-2α as therapeutic targets. However, genetic deletion of Hif-1α has no effect on mouse AML maintenance and may accelerate disease development. Here, we report the impact of conditional genetic deletion of Hif-2α or both Hif-1α and Hif-2α at different stages of leukemogenesis in mice. Deletion of Hif-2α accelerates development of leukemic stem cells (LSCs) and shortens AML latency initiated by Mll-AF9 and its downstream effectors Meis1 and Hoxa9. Notably, the accelerated initiation of AML caused by Hif-2α deletion is further potentiated by Hif-1α codeletion. However, established LSCs lacking Hif-2α or both Hif-1α and Hif-2α propagate AML with the same latency as wild-type LSCs. Furthermore, pharmacological inhibition of the HIF pathway or HIF-2α knockout using the lentiviral CRISPR-Cas9 system in human established leukemic cells with MLL-AF9 translocation have no impact on their functions. We therefore conclude that although Hif-1α and Hif-2α synergize to suppress the development of AML, they are not required for LSC maintenance. PMID:26642852

  16. Acute myeloid leukemia (AML) - children

    Science.gov (United States)

    Acute myeloid leukemia is a cancer of the blood and bone marrow. Bone marrow is the soft tissue inside ... develops quickly. Both adults and children can get acute myeloid leukemia ( AML ). This article is about AML in children.

  17. A stable transcription factor complex nucleated by oligomeric AML1–ETO controls leukaemogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Xiao-Jian; Wang, Zhanxin; Wang, Lan; Jiang, Yanwen; Kost, Nils; Soong, T. David; Chen, Wei-Yi; Tang, Zhanyun; Nakadai, Tomoyoshi; Elemento, Olivier; Fischle, Wolfgang; Melnick, Ari; Patel, Dinshaw J.; Nimer, Stephen D.; Roeder, Robert G.

    2013-06-30

    Transcription factors are frequently altered in leukaemia through chromosomal translocation, mutation or aberrant expression. AML1–ETO, a fusion protein generated by the t(8;21) translocation in acute myeloid leukaemia, is a transcription factor implicated in both gene repression and activation. AML1–ETO oligomerization, mediated by the NHR2 domain, is critical for leukaemogenesis, making it important to identify co-regulatory factors that ‘read’ the NHR2 oligomerization and contribute to leukaemogenesis. Here we show that, in human leukaemic cells, AML1–ETO resides in and functions through a stable AML1–ETO-containing transcription factor complex (AETFC) that contains several haematopoietic transcription (co)factors. These AETFC components stabilize the complex through multivalent interactions, provide multiple DNA-binding domains for diverse target genes, co-localize genome wide, cooperatively regulate gene expression, and contribute to leukaemogenesis. Within the AETFC complex, AML1–ETO oligomerization is required for a specific interaction between the oligomerized NHR2 domain and a novel NHR2-binding (N2B) motif in E proteins. Crystallographic analysis of the NHR2–N2B complex reveals a unique interaction pattern in which an N2B peptide makes direct contact with side chains of two NHR2 domains as a dimer, providing a novel model of how dimeric/oligomeric transcription factors create a new protein-binding interface through dimerization/oligomerization. Intriguingly, disruption of this interaction by point mutations abrogates AML1–ETO-induced haematopoietic stem/progenitor cell self-renewal and leukaemogenesis. These results reveal new mechanisms of action of AML1–ETO, and provide a potential therapeutic target in t(8;21)-positive acute myeloid leukaemia.

  18. The Hematopoietic Transcription Factors RUNX1 and ERG Prevent AML1-ETO Oncogene Overexpression and Onset of the Apoptosis Program in t(8;21) AMLs

    NARCIS (Netherlands)

    Mandoli, Amit; Singh, Abhishek A.; Prange, Koen H. M.; Tijchon, Esther; Oerlemans, Marjolein; Dirks, Rene; Ter Huurne, Menno; Wierenga, Albertus T. J.; Janssen-Megens, Eva M.; Berentsen, Kim; Sharifi, Nilofar; Kim, Bowon; Matarese, Filomena; Nguyen, Luan N.; Hubner, Nina C.; Rao, Nagesha A.; van den Akker, Emile; Altucci, Lucia; Vellenga, Edo; Stunnenberg, Hendrik G.; Martens, Joost H. A.

    2016-01-01

    The t(8;21) acute myeloid leukemia (AML)-associated oncoprotein AML1-ETO disrupts normal hematopoietic differentiation. Here, we have investigated its effects on the transcriptome and epigenome in t(8,21) patient cells. AML1-ETO binding was found at promoter regions of active genes with high levels

  19. An efficient protocol for the generation of monocyte derived dendritic cells using serum-free media for clinical applications in post remission AML patients.

    Science.gov (United States)

    da Silva Simoneti, Gisele; Saad, Sara Teresinha Olalla; Gilli, Simone Cristina Olenscki

    2014-01-01

    Protocols for the generation of dendritic cells (DCs) using serum as a supplementation of culture media leads to reactions due to animal proteins and disease transmissions. Several types of serum-free media (SFM), based on "good manufacture practices" (GMP), have recently been used and seem to be a viable option. The aim of this study was to evaluate the results of the differentiation, maturation, and function of DCs from Acute Myeloid Leukemia patients (AML), generated in SFM and medium supplemented with autologous serum (AS). DCs were analyzed by phenotype characteristics, viability, and functionality. The results showed the possibility of generating viable DCs in all the conditions tested. In patients, the X-VIVO 15 medium was more efficient than the other media tested in the generation of DCs producing IL-12p70 (p=0.05). Moreover, the presence of AS led to a significant increase of IL-10 by DCs as compared with CellGro (p=0.05) and X-Vivo15 (p=0.05) media, both in patients and donors. We concluded that SFM was efficient in the production of DCs for immunotherapy in AML patients. However, the use of AS appears to interfere with the functional capacity of the generated DCs.

  20. Normal karyotype mosaicism in adult AML patients with adverse-risk and undefined karyotype: preliminary report of treatment outcomes after hematopoietic stem cell transplantation.

    Science.gov (United States)

    Yoon, Jae-Ho; Kim, Hee-Je; Shin, Seung-Hwan; Yahng, Seung-Ah; Cho, Byung-Sik; Eom, Ki-Seong; Kim, Yoo-Jin; Lee, Seok; Min, Chang-Ki; Cho, Seok-Goo; Kim, Dong-Wook; Lee, Jong-Wook; Min, Woo-Sung; Park, Chong-Won

    2013-06-01

    Karyotype analysis in acute myeloid leukemia (AML) is one of the powerful prognostic factors for complete remission (CR), relapse, and overall survival (OS). Cytogenetic mosaicism is considered to be one of the important characteristics in expression of phenotypic manifestations. However, it has not come into focus due to emerging molecular biological approaches and the results of a number of mutation studies. Clinical correlates and prognostic relevance of mosaicism were evaluated in 163 AML patients [adverse-risk karyotypes (n = 72) and undefined karyotypes (n = 91)]. All patients were treated by induction and consolidation chemotherapies and finally went on hematopoietic stem cell transplantations (HSCT). Patients were divided into two subgroups, either with or without normal karyotype (NK) mosaicism. Seventy patients exhibited NK mosaicism and 93 did not. There were no significant differences in age, gender, chemotherapy cycles to achieve CR, HSCT donor type, source or intensity properties between the two subgroups. We found that NK mosaicism remaining in adverse-risk and undefined karyotype at diagnosis significantly correlates with better OS (p = 0.001) and lower CIR (p = 0.021) rate after HSCT. Our data show that the poor prognostic properties of unfavorable risk karyotype can be overcome to a great extent by allogeneic HSCT and chronic GVHD, especially in the subgroup with NK mosaicism. Cytogenetic mosaicism at initial diagnosis can be an influential factor for survival outcomes, even after HSCT.

  1. Nucleosome Presence at AML-1 Binding Sites Inversely Correlates with Ly49 Expression: Revelations from an Informatics Analysis of Nucleosomes and Immune Cell Transcription Factors.

    Science.gov (United States)

    Wight, Andrew; Yang, Doo; Ioshikhes, Ilya; Makrigiannis, Andrew P

    2016-04-01

    Beyond its role in genomic organization and compaction, the nucleosome is believed to participate in the regulation of gene transcription. Here, we report a computational method to evaluate the nucleosome sensitivity for a transcription factor over a given stretch of the genome. Sensitive factors are predicted to be those with binding sites preferentially contained within nucleosome boundaries and lacking 10 bp periodicity. Based on these criteria, the Acute Myeloid Leukemia-1a (AML-1a) transcription factor, a regulator of immune gene expression, was identified as potentially sensitive to nucleosomal regulation within the mouse Ly49 gene family. This result was confirmed in RMA, a cell line with natural expression of Ly49, using MNase-Seq to generate a nucleosome map of chromosome 6, where the Ly49 gene family is located. Analysis of this map revealed a specific depletion of nucleosomes at AML-1a binding sites in the expressed Ly49A when compared to the other, silent Ly49 genes. Our data suggest that nucleosome-based regulation contributes to the expression of Ly49 genes, and we propose that this method of predicting nucleosome sensitivity could aid in dissecting the regulatory role of nucleosomes in general.

  2. Switching CAR T cells on and off: a novel modular platform for retargeting of T cells to AML blasts

    Science.gov (United States)

    Cartellieri, M; Feldmann, A; Koristka, S; Arndt, C; Loff, S; Ehninger, A; von Bonin, M; Bejestani, E P; Ehninger, G; Bachmann, M P

    2016-01-01

    The adoptive transfer of CD19-specific chimeric antigen receptor engineered T cells (CAR T cells) resulted in encouraging clinical trials in indolent B-cell malignancies. However, they also show the limitations of this fascinating technology: CAR T cells can lead to even life-threatening off-tumor, on-target side effects if CAR T cells crossreact with healthy tissues. Here, we describe a novel modular universal CAR platform technology termed UniCAR that reduces the risk of on-target side effects by a rapid and reversible control of CAR T-cell reactivity. The UniCAR system consists of two components: (1) a CAR for an inert manipulation of T cells and (2) specific targeting modules (TMs) for redirecting UniCAR T cells in an individualized time- and target-dependent manner. UniCAR T cells can be armed against different tumor targets simply by replacement of the respective TM for (1) targeting more than one antigen simultaneously or subsequently to enhance efficacy and (2) reducing the risk for development of antigen-loss tumor variants under treatment. Here we provide ‘proof of concept' for retargeting of UniCAR T cells to CD33- and/or CD123-positive acute myeloid leukemia blasts in vitro and in vivo. PMID:27518241

  3. A functional study on the migration of human monocytes to human leukemic cell lines and the role of monocyte chemoattractant protein-1

    NARCIS (Netherlands)

    Legdeur, MCJC; Beelen, RHJ; Schuurhuis, GJ; Broekhoven, MG; vandeLoosdrecht, AA; Tekstra, J; Langenhuijsen, MMAC; Ossenkoppele, GJ

    1997-01-01

    In the present study the migration of human monocytes towards the supernatants of five different human myeloid leukemic cell lines, four different human lymphatic leukemic cell lines and blasts derived from three different patients with acute myeloid leukemia (AML) was studied and the role of monocy

  4. AML Guide: Information for Patients and Caregivers

    Science.gov (United States)

    ... be used to identify the type of AML cells. Immunotherapy. The treatments that are used to boost the body’s immune system. Leukemia. A cancer of the marrow and blood. Lymph nodes. Small bean-shaped organs around the body that are ...

  5. Azacitidine in AML: a treatment option?

    NARCIS (Netherlands)

    Huls, G.A.

    2015-01-01

    In this issue of Blood, Dombret et al report the final analysis of the international phase 3 study of azacitidine vs conventional care regimens in older (>/=65 years), newly diagnosed acute myeloid leukemia (AML) patients with >30% bone marrow blasts and white blood cell (WBC) counts

  6. The role of C/EBPa in PD-1+ CD4+ T cells & modulation of RNR activity prolongs survival of mice with AML

    DEFF Research Database (Denmark)

    Norrie, Ida Christine

    The Ph.D. thesis comprises two projects: (1) C/EBPα is dispensable for the ontogeny of PD1+ CD4+ memory T cells, but restricts their expansion in an age-dependent manner. (2) Modulation of RNR activity prolongs survival of mice with AML. Immunosenescence is a condition of the immune system...... the de novo synthesis of deoxyribonucleotides (dNTPs) to provide a strictly balanced supply of dNTPs required for DNA synthesis and DNA repair. The activity of this enzyme is under exquisite transcriptional and posttranscriptional control to avoid unbalanced dNTP pools that can lead to mis...... to DNA damage, and its activity by allosteric regulation by nucleoside triphosphates and inhibitory proteins. In S. cerevisiae, Sml1 and Dif1 have been shown to bind to RNR and inhibit its activity, whereas in S. pombe the inhibitory protein is Spd1. It has been demonstrated that the inability to degrade...

  7. Autologous stem cell transplantation after complete remission and first consolidation in acute myeloid leukemia patients aged 61-70 years: results of the prospective EORTC-GIMEMA AML-13 study.

    NARCIS (Netherlands)

    Thomas, X.; Suciu, S.; Rio, B.; Leone, G.; Broccia, G.; Fillet, G.; Jehn, U.; Feremans, W.; Meloni, G.; Vignetti, M.; Witte, T.J.M. de; Amadori, S.

    2007-01-01

    BACKGROUND AND OBJECTIVES: The optimal post-remission treatment for elderly patients with acute myeloid leukemia (AML) is presently unknown. Recent studies have reported the feasibility of autologous peripheral blood stem cell transplantation (PBSCT) in this population. We evaluate the outcome of th

  8. Oridonin effectively reverses the drug resistance of cisplatin involving induction of cell apoptosis and inhibition of MMP expression in human acute myeloid leukemia cells

    Directory of Open Access Journals (Sweden)

    Yuan Zhang

    2017-03-01

    Full Text Available Cisplatin is the first generation platinum-based chemotherapy agent. However, the extensive application of cisplatin inevitably causes drug resistance, which is a major obstacle to cancer chemotherapy. Oridonin is a diterpenoid isolated from Rabdosia rubescens with potent anticancer activity. The aim of our study is to investigate the role of oridonin to reverse the cisplatin-resistance in human acute myeloid leukemia (AML cells. The effect of oridonin on human AML cell proliferation was evaluated by MTT assay, cell migration and invasion were evaluated by transwell migration and invasion assays in cisplatin-resistant human AML cells. Furthermore, cell apoptosis was examined by flow cytometry. The inhibitive effect of oridonin in vivo was determined using xenografted nude mice. In addition, the expressions of MMP2 and MMP9 were detected by Western blot. There was a synergistic antitumor effect between cisplatin and oridonin on cisplatin-resistant human AML cells in vitro and in vivo. In addition, the combination of cisplatin and oridonin synergistically induced cell apoptosis. Furthermore, the combination treatment not only inhibited AML cell migration and invasion, but more significantly, decreased the expressions of MMP2 and MMP9 proteins. Our results suggest that the synergistic effect between both agents is likely to be driven by the inhibition of MMP expression and the resulting increased apoptosis.

  9. Monitoring AML1-ETO mRNA levels by real-time quantitative RT-PCR in t (8; 21) acute myeloid leukemia patients after hematopoietic stem cell transplantation%实时定量RT-PCR方法监测急性髓系白血病患者造血干细胞移植后AML1-ETO融合基因mRNA水平的临床意义

    Institute of Scientific and Technical Information of China (English)

    王志东; 秦亚溱; 刘艳荣; 许兰平; 刘代红; 刘开彦; 黄晓军

    2008-01-01

    目的 评价实时定量RT-PCR(Q-PCR)方法监测AML1-ETO(+)急性髓系白血病(AML)患者异基因造血干细胞移植(allo-HSCT)后AML1-ETO mRNA水平的表达及其临床意义.方法 采用基于TagMan探针的Q-PCR技术检测17例AML1-ETO(+)AML患者allo-HSCT后不同时间骨髓标本AML1-ETO mRNA的表达.AML1-ETO mRNA水平以内参基因abl进行归一化.采用荧光原位杂交(FISH)法评估HSCT后是否达到细胞遗传学完全缓解(CCyR).结果 Q-PCR实验可重复敏感度为5个拷贝.在16例CCyR患者中,1例死于移植物抗宿主病(GVHD),1例死于感染,其余14例中位随访时间为268(70~811)d,HSCT后1个月(+1月)AML1-ETO中位水平0(0~0.740),+2月为0.026(0~2.900),+3月为0.039(0~3.300).移植时间超过12个月的5例患者中,中位随访685(385~811)d,4例仍呈AML1-ETO阳性,中位值0.078(0.003~0.120).1例复发患者+1月为0,+2月为9.800,+3月为5.600,+110 d血液学复发,AML1-ETO mRNA为390.000,+382 d死亡.结论 1年内AML1-ETO持续低水平阳性不一定预示复发;对AML1-ETO(+)AML患者HSCT后定期动态监测AML1-ETO水平十分必要.%Objective To evaluate the value of real time quantitative RT-PCR(Q-PCR) for monitoring AML1-ETO mRNA levels in AMLI-ETO(+) acute myeloid leukemia (AML) patients following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods Quantification of AMLI-ETO (+) mRNA was performed serially on bone marrow samples from 17 patients with AML1-ETO (+) AML after HSCT. Q-PCR used the TagMan probe system. The AML1-ETO mRNA level was normalized by control gene abl. Cytogenetic response was evaluated by fluorescent in situ hybridization (FISH). Results The reproducible sensitivity of Q-PCR was 5 copies. Out of 16 patients who achieved sustained complete cytogenetic response (CCyR), one each died of graft-versus-host disease and infection. The median AML1-ETO mRNA levels in the rest of 14 CCyR patients were 0 (0 - 0.740), 0. 026 (0 - 2.900), 0.039 (0 - 3.300) at

  10. SGN-CD33A: a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML.

    Science.gov (United States)

    Kung Sutherland, May S; Walter, Roland B; Jeffrey, Scott C; Burke, Patrick J; Yu, Changpu; Kostner, Heather; Stone, Ivan; Ryan, Maureen C; Sussman, Django; Lyon, Robert P; Zeng, Weiping; Harrington, Kimberly H; Klussman, Kerry; Westendorf, Lori; Meyer, David; Bernstein, Irwin D; Senter, Peter D; Benjamin, Dennis R; Drachman, Jonathan G; McEarchern, Julie A

    2013-08-22

    Outcomes in acute myeloid leukemia (AML) remain unsatisfactory, and novel treatments are urgently needed. One strategy explores antibodies and their drug conjugates, particularly those targeting CD33. Emerging data with gemtuzumab ozogamicin (GO) demonstrate target validity and activity in some patients with AML, but efficacy is limited by heterogeneous drug conjugation, linker instability, and a high incidence of multidrug resistance. We describe here the development of SGN-CD33A, a humanized anti-CD33 antibody with engineered cysteines conjugated to a highly potent, synthetic DNA cross-linking pyrrolobenzodiazepine dimer via a protease-cleavable linker. The use of engineered cysteine residues at the sites of drug linker attachment results in a drug loading of approximately 2 pyrrolobenzodiazepine dimers per antibody. In preclinical testing, SGN-CD33A is more potent than GO against a panel of AML cell lines and primary AML cells in vitro and in xenotransplantation studies in mice. Unlike GO, antileukemic activity is observed with SGN-CD33A in AML models with the multidrug-resistant phenotype. Mechanistic studies indicate that the cytotoxic effects of SGN-CD33A involve DNA damage with ensuing cell cycle arrest and apoptotic cell death. Together, these data suggest that SGN-CD33A has CD33-directed antitumor activity and support clinical testing of this novel therapeutic in patients with AML.

  11. KEGG DISEASE / Acute myeloid leukemia (AML) [KEGG DISEASE

    Lifescience Database Archive (English)

    Full Text Available DISEASE: H00003 Entry H00003Disease Name Acute myeloid leukemia (AML) Description Acute.... Category Cancer Brite Human diseases [BR:br08402] Cancers Cancers of haematopoietic and lymphoid tissues H00003Acute...atopoietic and related tissue C92Myeloid leukaemia H00003Acute myeloid leukemia (AML) Cancer-accociated carb...ohydrates [br08441.html] H00003 Pathway hsa05221Acute myeloid leukemiahsa05202Transcriptional misregulation ... or t(16; 16)(p13, q22), (CBF-beta/MYH11) ICD-O: 9866/3, Tumor type: Acute promyelocytic leukaemia (AML with

  12. Killer Ig-like receptor (KIR) compatibility plays a role in the prevalence of acute GVHD in unrelated hematopoietic cell transplants for AML.

    Science.gov (United States)

    Sun, J Y; Gaidulis, L; Dagis, A; Palmer, J; Rodriguez, R; Miller, M M; Forman, S J; Senitzer, D

    2005-09-01

    Killer Ig-like receptor (KIR) is a major cluster of the natural killer cell receptors and may play a role in the outcome of hematopoietic cell transplants. A total of 65 AML cases transplanted with T-replete hematopoietic cells from unrelated donors were retrospectively KIR-genotyped by a multiplex PCR method of our own design. The KIR gene frequency and genotype patterns in these 130 samples were consistent with the data in the literature. Based upon overall inhibitory and activating KIR genes in both donors and patients, we developed an algorithm to calculate a compatibility score for each transplant case as plus, zero or minus. Significantly higher incidence (18/20, 90%) of acute (a) GVHD (grade II-IV) was found in the transplant cases with plus scores than that (25/45, 56%) in the cases with zero or minus scores (P compatibility calculated by this algorithm may predict aGVHD incidence and be helpful in choosing donors.

  13. Reasons for treating secondary AML as de novo AML

    DEFF Research Database (Denmark)

    Ostgård, Lene Sofie Granfeldt; Kjeldsen, Eigil; Holm, Mette Skov;

    2010-01-01

    ) in 77 cases (49%), CMPD (chronic myeloproliferative disorder) in 43 cases (27%) and was therapy-related AML (t-AML) in 37 cases (24%). Median age at diagnosis of AML was 69 yr in secondary cases when compared to 66 yr in de novo cases (P = 0.006). In univariate analyses, secondary AML was associated...... with an inferior complete remission (CR) rate (P = 0.008) and poorer overall survival (OS, P = 0.003) whereas in complete remitters, disease-free survival (DFS) of secondary cases was equal to that of de novo cases. Interestingly, in all further analyses of CR-rates, OS and DFS, when correcting for the influence...

  14. Generating Peripheral Blood Derived Lymphocytes Reacting Against Autologous Primary AML Blasts

    OpenAIRE

    Mehta, Rohtesh S.; Chen, Xiaohua; Antony, Jeyaraj; Boyiadzis, Michael; Szabolcs, Paul

    2016-01-01

    Expanding on our prior studies with cord blood T-cells, we hypothesized that primary AML-reactive autologous T-cells could be generated ex vivo under immunomodulatory conditions. We purified AML and T-cells from 8 newly diagnosed high-risk patients. After 2 weeks expansion, T-cells were stimulated with IFN-γ treated autologous AML weekly X 3, IL-15 and agonistic anti-CD28 antibody. CTL and ELISpot assays tested functionality; RT-qPCR tested AML and T-cell gene expression profiles. Based on co...

  15. Downregulation but lack of promoter hypermethylation or somatic mutations of the potential tumor suppressor CXXC5 in MDS and AML with deletion 5q

    DEFF Research Database (Denmark)

    Treppendahl, Marianne Bach; Möllgård, L; Hellström-Lindberg, E

    2013-01-01

    During recent years mutations in epigenetic modulators have been identified in several human cancers, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS)[1]. CXXC5 has been found to be necessary for retinoic acid induced differentiation of myelocytic leukemia cells, identify......During recent years mutations in epigenetic modulators have been identified in several human cancers, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS)[1]. CXXC5 has been found to be necessary for retinoic acid induced differentiation of myelocytic leukemia cells...

  16. EVI1 is critical for the pathogenesis of a subset of MLL-AF9-rearranged AMLs.

    Science.gov (United States)

    Bindels, Eric M J; Havermans, Marije; Lugthart, Sanne; Erpelinck, Claudia; Wocjtowicz, Elizabeth; Krivtsov, Andrei V; Rombouts, Elwin; Armstrong, Scott A; Taskesen, Erdogan; Haanstra, Jurgen R; Beverloo, H Berna; Döhner, Hartmut; Hudson, Wendy A; Kersey, John H; Delwel, Ruud; Kumar, Ashish R

    2012-06-14

    The proto-oncogene EVI1 (ecotropic viral integration site-1), located on chromosome band 3q26, is aberrantly expressed in human acute myeloid leukemia (AML) with 3q26 rearrangements. In the current study, we showed, in a large AML cohort carrying 11q23 translocations, that ∼ 43% of all mixed lineage leukemia (MLL)-rearranged leukemias are EVI1(pos). High EVI1 expression occurs in AMLs expressing the MLL-AF6, -AF9, -AF10, -ENL, or -ELL fusion genes. In addition, we present evidence that EVI1(pos) MLL-rearranged AMLs differ molecularly, morphologically, and immunophenotypically from EVI1(neg) MLL-rearranged leukemias. In mouse bone marrow cells transduced with MLL-AF9, we show that MLL-AF9 fusion protein maintains Evi1 expression on transformation of Evi1(pos) HSCs. MLL-AF9 does not activate Evi1 expression in MLL-AF9-transformed granulocyte macrophage progenitors (GMPs) that were initially Evi1(neg). Moreover, shRNA-mediated knockdown of Evi1 in an Evi1(pos) MLL-AF9 mouse model inhibits leukemia growth both in vitro and in vivo, suggesting that Evi1 provides a growth-promoting signal. Using the Evi1(pos) MLL-AF9 mouse leukemia model, we demonstrate increased sensitivity to chemotherapeutic agents on reduction of Evi1 expression. We conclude that EVI1 is a critical player in tumor growth in a subset of MLL-rearranged AMLs.

  17. Cell differentiation mediated by co-culture of human umbilical cord blood stem cells with murine hepatic cells.

    Science.gov (United States)

    Stecklum, Maria; Wulf-Goldenberg, Annika; Purfürst, Bettina; Siegert, Antje; Keil, Marlen; Eckert, Klaus; Fichtner, Iduna

    2015-02-01

    In the present study, purified human cord blood stem cells were co-cultivated with murine hepatic alpha mouse liver 12 (AML12) cells to compare the effect on endodermal stem cell differentiation by either direct cell-cell interaction or by soluble factors in conditioned hepatic cell medium. With that approach, we want to mimic in vitro the situation of preclinical transplantation experiments using human cells in mice. Cord blood stem cells, cultivated with hepatic conditioned medium, showed a low endodermal differentiation but an increased connexin 32 (Cx32) and Cx43, and cytokeratin 8 (CK8) and CK19 expression was monitored by reverse transcription polymerase chain reaction (RT-PCR). Microarray profiling indicated that in cultivated cord blood cells, 604 genes were upregulated 2-fold, with the highest expression for epithelial CK19 and epithelial cadherin (E-cadherin). On ultrastructural level, there were no major changes in the cellular morphology, except a higher presence of phago(ly)some-like structures observed. Direct co-culture of AML12 cells with cord blood cells led to less incisive differentiation with increased sex-determining region Y-box 17 (SOX17), Cx32 and Cx43, as well as epithelial CK8 and CK19 expressions. On ultrastructural level, tight cell contacts along the plasma membranes were revealed. FACS analysis in co-cultivated cells quantified dye exchange on low level, as also proved by time relapse video-imaging of labelled cells. Modulators of gap junction formation influenced dye transfer between the co-cultured cells, whereby retinoic acid increased and 3-heptanol reduced the dye transfer. The study indicated that the cell-co-cultured model of human umbilical cord blood cells and murine AML12 cells may be a suitable approach to study some aspects of endodermal/hepatic cell differentiation induction.

  18. Modified EBMT Pretransplant Risk Score Can Identify Favorable-risk Patients Undergoing Allogeneic Hematopoietic Cell Transplantation for AML, Not Identified by the HCT-CI Score.

    Science.gov (United States)

    Michelis, Fotios V; Messner, Hans A; Uhm, Jieun; Alam, Naheed; Lambie, Anna; McGillis, Laura; Seftel, Matthew D; Gupta, Vikas; Kuruvilla, John; Lipton, Jeffrey H; Kim, Dennis Dong Hwan

    2015-05-01

    Risk scores have been developed for allogeneic hematopoietic cell transplantation (HCT) outcomes, such as the Hematopoietic Cell Transplantation-Comorbidity Index (HCT-CI) and the modified European Group for Blood and Marrow Transplantation risk score (mEBMT) for acute leukemia. We investigated the influence of these scores for 350 patients who underwent transplantation for acute myeloid leukemia (AML). The HCT-CI scores were grouped as 0 to 2 and ≥ 3 (231 and 119 patients, respectively) and the mEBMT scores as 0 to 2 and ≥ 3 (166 and 184 patients, respectively). Univariate analysis showed a significant association between the HCT-CI score and overall survival (OS) (P = .01), as did the mEBMT score (P = .002). The 5-year OS rate was 50% and 34% for a mEBMT score of 0 to 2 and ≥ 3, respectively. A subgroup of patients with a mEBMT score of 0 to 1 (n = 32) demonstrated a favorable OS of 75% at 5 years. This subgroup was younger (median age, 31 years), in first remission at transplantation, and had related donors. For the HCT-CI, the 5-year OS was 46% and 34% for a score of 0 to 2 and ≥ 3, respectively. Patients with an HCT-CI score of 0 (n = 94) had a 5-year OS of 44%. Multivariable analysis confirmed both the HCT-CI score and the mEBMT score, as previously grouped, as independent prognostic variables for both OS (P = .02 and P = .001, respectively) and nonrelapse mortality (NRM) (P = .01 and P = .003, respectively). The results of the present study have demonstrated that the HCT-CI and mEBMT are both prognostic for OS and NRM in our cohort. However, the mEBMT score can identify a favorable-risk subgroup of patients not identifiable using the HCT-CI. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. A Rapid Culture Technique Produces Functional Dendritic-Like Cells from Human Acute Myeloid Leukemia Cell Lines

    Directory of Open Access Journals (Sweden)

    Jian Ning

    2011-01-01

    Full Text Available Most anti-cancer immunotherapeutic strategies involving dendritic cells (DC as vaccines rely upon the adoptive transfer of DC loaded with exogenous tumour-peptides. This study utilized human acute myeloid leukemia (AML cells as progenitors from which functional dendritic-like antigen presenting cells (DLC were generated, that constitutively express tumour antigens for recognition by CD8+ T cells. DLC were generated from AML cell lines KG-1 and MUTZ-3 using rapid culture techniques and appropriate cytokines. DLC were evaluated for their cell-surface phenotype, antigen uptake and ability to stimulate allogeneic responder cell proliferation, and production of IFN-γ; compared with DC derived from normal human PBMC donors. KG-1 and MUTZ-3 DLC increased expression of CD80, CD83, CD86, and HLA-DR, and MUTZ-3 DLC downregulated CD14 and expressed CD1a. Importantly, both KG-1 and MUTZ-3-derived DLC promoted proliferation of allogeneic responder cells more efficiently than unmodified cells; neither cells incorporated FITC-labeled dextran, but both stimulated IFN-γ production from responding allogeneic CD8+ T cells. Control DC produced from PBMC using the FastDC culture also expressed high levels of critical cell surface ligands and demonstrated good APC function. This paper indicates that functional DLC can be cultured from the AML cell lines KG-1 and MUTZ-3, and FastDC culture generates functional KG-1 DLC.

  20. Hypomethylation and up-regulation of PD-1 in T cells by azacytidine in MDS/AML patients

    DEFF Research Database (Denmark)

    Ørskov, Andreas D; Treppendahl, Marianne B; Skovbo, Anni

    2015-01-01

    The hypomethylating agents (HMAs) are standard therapy for patients with higher-risk myelodysplastic syndrome (MDS); however, the majority of the patients will lose their response to HMAs over time due to unknown mechanisms. It has recently been shown that T cell expression of the immunoinhibitory......-responding patients compared to healthy controls (p = 0.023). Accordingly, in addition to their beneficial function, HMAs induce PD-1 expression on T cells in the MDS microenvironment, thereby likely hampering the immune response against the MDS blasts. Thus, we suggest that activation of the PD-1 checkpoint during...

  1. Regulation of Trib2 by an E2F1-C/EBPalpha feedback loop in AML cell proliferation

    DEFF Research Database (Denmark)

    Rishi, Loveena; Hannon, Maura; Salomè, Mara;

    2014-01-01

    The loss of regulation of cell proliferation is a key event in leukemic transformation, and the oncogene Trib2 is emerging as a pivotal target of transcription factors in acute leukemias. Deregulation of the transcription factor E2F1, normally repressed by C/EBPalpha-p42, occurs in acute myeloid ...

  2. Generating Peripheral Blood Derived Lymphocytes Reacting Against Autologous Primary AML Blasts

    Science.gov (United States)

    Mehta, Rohtesh S.; Chen, Xiaohua; Antony, Jeyaraj; Boyiadzis, Michael; Szabolcs, Paul

    2015-01-01

    Expanding on our prior studies with cord blood T-cells, we hypothesized that primary AML-reactive autologous T-cells could be generated ex vivo under immunomodulatory conditions. We purified AML and T-cells from 8 newly diagnosed high-risk patients. After 2 weeks expansion, T-cells were stimulated with IFN-γ treated autologous AML weekly X 3, IL-15 and agonistic anti-CD28 antibody. CTL and ELISpot assays tested functionality; RT-qPCR tested AML and T-cell gene expression profiles. Based on combined positive ELIspot and CTL assays, T-cells reactive against AML were generated in 5/8 patients. Treg proportion declined post-co-cultures in reactive T-cell samples. AML-reactive T-cells displayed an activated gene expression profile. “Resistant” AML blasts displayed genes associated with immunosuppressive MDSC. We discuss our approach to creating primary AML-reactive autologous T-cell and limitations that require further work. Our study provides a platform for future research targeting on generating autologous leukemia reactive T-cells. PMID:26849076

  3. A robust and rapid xenograft model to assess efficacy of chemotherapeutic agents for human acute myeloid leukemia.

    Science.gov (United States)

    Saland, E; Boutzen, H; Castellano, R; Pouyet, L; Griessinger, E; Larrue, C; de Toni, F; Scotland, S; David, M; Danet-Desnoyers, G; Vergez, F; Barreira, Y; Collette, Y; Récher, C; Sarry, J-E

    2015-03-20

    Relevant preclinical mouse models are crucial to screen new therapeutic agents for acute myeloid leukemia (AML). Current in vivo models based on the use of patient samples are not easy to establish and manipulate in the laboratory. Our objective was to develop robust xenograft models of human AML using well-characterized cell lines as a more accessible and faster alternative to those incorporating the use of patient-derived AML cells. Five widely used AML cell lines representing various AML subtypes were transplanted and expanded into highly immunodeficient non-obese diabetic/LtSz-severe combined immunodeficiency IL2Rγc(null) mice (for example, cell line-derived xenografts). We show here that bone marrow sublethal conditioning with busulfan or irradiation has equal efficiency for the xenotransplantation of AML cell lines. Although higher number of injected AML cells did not change tumor engraftment in bone marrow and spleen, it significantly reduced the overall survival in mice for all tested AML cell lines. On the basis of AML cell characteristics, these models also exhibited a broad range of overall mouse survival, engraftment, tissue infiltration and aggressiveness. Thus, we have established a robust, rapid and straightforward in vivo model based on engraftment behavior of AML cell lines, all vital prerequisites for testing new therapeutic agents in preclinical studies.

  4. Recombinant human IL-3 and G-CSF act synergistically in stimulating the growth of acute myeloid leukemia cells.

    Science.gov (United States)

    Pébusque, M J; Faÿ, C; Lafage, M; Sempéré, C; Saeland, S; Caux, C; Mannoni, P

    1989-03-01

    The effects of combinations of recombinant human growth factors (colony-stimulating factor (CSF], interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony stimulating factor (G-CSF) for inducing proliferation of leukemic cells were compared in 27 acute myeloid leukemias (AMLs). While functional heterogeneity of AML was clearly shown, we further demonstrated that optimal growth may be obtained with combinations of CSF. The most striking feature was that, in both suspension and semisolid cultures, IL-3 and G-CSF acted synergistically in supporting AML cell proliferation except in cases for which G-CSF was found to be an inhibitory factor. In the majority of cases, the proliferative effects of the IL-3 and GM-CSF combination were significantly higher than the most potent of either factor present alone in the cultures. Finally, preincubation with IL-3 greatly potentiated the responsiveness of AML cells to subsequent addition of either GM-CSF or G-CSF. These results indicate that AML cells respond to growth factor in the same way as normal hemopoietic cells and that stimulation by a second late-acting growth factor such as G-CSF is also required to yield optimal growth.

  5. CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia.

    Science.gov (United States)

    Kenderian, S S; Ruella, M; Shestova, O; Klichinsky, M; Aikawa, V; Morrissette, J J D; Scholler, J; Song, D; Porter, D L; Carroll, M; June, C H; Gill, S

    2015-08-01

    Patients with chemo-refractory acute myeloid leukemia (AML) have a dismal prognosis. Chimeric antigen receptor T (CART) cell therapy has produced exciting results in CD19+ malignancies and may overcome many of the limitations of conventional leukemia therapies. We developed CART cells to target CD33 (CART33) using the anti-CD33 single chain variable fragment used in gemtuzumab ozogamicin (clone My96) and tested the activity and toxicity of these cells. CART33 exhibited significant effector functions in vitro and resulted in eradication of leukemia and prolonged survival in AML xenografts. CART33 also resulted in human lineage cytopenias and reduction of myeloid progenitors in xenograft models of hematopoietic toxicity, suggesting that permanently expressed CD33-specific CART cells would have unacceptable toxicity. To enhance the viability of CART33 as an option for AML, we designed a transiently expressed mRNA anti-CD33 CAR. Gene transfer was carried out by electroporation into T cells and resulted in high-level expression with potent but self-limited activity against AML. Thus our preclinical studies show potent activity of CART33 and indicate that transient expression of anti-CD33 CAR by RNA modification could be used in patients to avoid long-term myelosuppression. CART33 therapy could be used alone or as part of a preparative regimen prior to allogeneic transplantation in refractory AML.

  6. Synergistic Effect of Decitabine and Valproic Acid on Differentiation and Apoptosts of Stem Cells of an AML - M2 Patient in vitro%地西他滨联合丙戊酸钠体外诱导1例AML-M2复发患者原始细胞凋亡分化的作用研究

    Institute of Scientific and Technical Information of China (English)

    陈琼娜; 王晔恺; 周吉航; 李翊卫; 曾芳; 刘晓光

    2012-01-01

    Objective To investigate the synergistic effect of decitabine( DCA) and valproic acid( VPA) on differentiation and apoptosis of stem cells of an AML - M2 patient in vitro. Methods The stem cells in the bone marrow of the AML - M2 patient were sorted by flow cytometry. The groups were set as follows:control group; DCA alone group A(1.0μmol/L); DCA alone group B(4. 0μmol/L); VPA alone group (2. 0mmol/L); combination group A (DCA l.0μmol/L + VPA 2. 0mmol/L); combination group B(DCA 4. 0μmol/L + VPA 2. 0mmol/L). The cells were treated by drug for 48 hours. Then the apoptosis rates,CD117 and CD14 expressions were detected by flow cytometry. Results Compared with corresponding concentration single drug group,the apoptosis rates and CD 14 expressions of the combination group A and B were significantly higher(P < 0. 01 ) and CD117 expressions of the combination group A and B were significantly lower( P < 0.01). Conclusion There was an enhanced antileukemia activity of combinations of DCA and VPA.%目的 探讨地西他滨( decitabine,DCA)和丙戊酸钠(valproic acid,VPA)联用对1例复发性AML - M2患者原始细胞体外的影响.方法 分选此例患者骨髓原始细胞,设立药物分组如下:对照组,DCA单药A组(1.0μmol/L),DCA单药B组(4.0μmol/L),VPA单药组(2.0mmol/L),联合用药A组(DCA 1.0μmol/L+ VPA 2.0mmol/L),联合用药B组(DCA 4.0μmol/L+VPA 2.0mmol/L),作用48h.应用流式细胞术检测早期凋亡率和CD117、CD14表达率.结果 相对于各自的单药组,联合用药A组和联合用药B组均能显著提高早期凋亡率和CD14表达,抑制CD117的表达(P<0.01).结论 体外DCA联合VPA能显著加强抗白血病效应.

  7. AML (Logistics Center) Local Area Network -

    Data.gov (United States)

    Department of Transportation — The AML LAN is designed to facilitate the services and resources needed to support the operations of the FAA Logistics Center users. The AML LAN provides support for...

  8. Securinine, a myeloid differentiation agent with therapeutic potential for AML.

    Directory of Open Access Journals (Sweden)

    Kalpana Gupta

    Full Text Available As the defining feature of Acute Myeloid Leukemia (AML is a maturation arrest, a highly desirable therapeutic strategy is to induce leukemic cell maturation. This therapeutic strategy has the potential of avoiding the significant side effects that occur with the traditional AML therapeutics. We identified a natural compound securinine, as a leukemia differentiation-inducing agent. Securinine is a plant-derived alkaloid that has previously been used clinically as a therapeutic for primarily neurological related diseases. Securinine induces monocytic differentiation of a wide range of myeloid leukemia cell lines as well as primary leukemic patient samples. Securinine's clinical potential for AML can be seen from its ability to induce significant growth arrest in cell lines and patient samples as well as its activity in significantly impairing the growth of AML tumors in nude mice. In addition, securinine can synergize with currently employed agents such as ATRA and decitabine to induce differentiation. This study has revealed securinine induces differentiation through the activation of DNA damage signaling. Securinine is a promising new monocytic differentiation inducing agent for AML that has seen previous clinical use for non-related disorders.

  9. Hyperdiploidy in Childhood AML Associated with Low Age and AML-M7. A NOPHO-AML Study and Literature Review

    DEFF Research Database (Denmark)

    Sandahl, Julie Damgaard; Abrahamson, Jonas; Heldrup, Jesper

    Introduction: High hyperdiploidy (51–67 chromosomes) is one of the most common cytogenetic abnormality patterns in childhood B-cell precursor acute lymphoblastic leukemia (ALL) and the clinical and cytogenetic features are well characterized. High hyperdiploidy in pediatric acute myeloid leukemia...... abnormalities among the pediatric hyperdiploid AML patients is relatively unknown. Current knowledge in this field is based on small adult series or case reports. In this descriptive study we describe the clinical - and cytogenetic features in childhood hyperdiploid AML with a modal chromosome number at 50...

  10. In Vivo Expansion of Co-Transplanted T Cells Impacts on Tumor Re-Initiating Activity of Human Acute Myeloid Leukemia in NSG Mice

    NARCIS (Netherlands)

    M. von Bonin (Malte); M. Wermke (Martin); K.N. Cosgun (Kadriye Nehir); C. Thiede; M. Bornhäuser (Martin); G. Wagemaker (Gerard); C. Waskow (Claudia)

    2013-01-01

    textabstractHuman cells from acute myeloid leukemia (AML) patients are frequently transplanted into immune-compromised mouse strains to provide an in vivo environment for studies on the biology of the disease. Since frequencies of leukemia re-initiating cells are low and a unique cell surface

  11. Clinical Results of Hypomethylating Agents in AML Treatment

    Directory of Open Access Journals (Sweden)

    Marjan Cruijsen

    2014-12-01

    Full Text Available Epigenetic changes play an important role in the development of acute myeloid leukemia (AML. Unlike gene mutations, epigenetic changes are potentially reversible, which makes them attractive for therapeutic intervention. Agents that affect epigenetics are the DNA methyltransferase inhibitors, azacitidine and decitabine. Because of their relatively mild side effects, azacitidine and decitabine are particularly feasible for the treatment of older patients and patients with co-morbidities. Both drugs have remarkable activity against AML blasts with unfavorable cytogenetic characteristics. Recent phase 3 trials have shown the superiority of azacitidine and decitabine compared with conventional care for older AML patients (not eligible for intensive treatment. Results of treatment with modifications of the standard azacitidine (seven days 75 mg/m2 SC; every four weeks and decitabine (five days 20 mg/m2 IV; every four weeks schedules have been reported. Particularly, the results of the 10-day decitabine schedule are promising, revealing complete remission (CR rates around 45% (CR + CRi (i.e., CR with incomplete blood count recovery around 64% almost comparable with intensive chemotherapy. Application of hypomethylating agents to control AML at the cost of minimal toxicity is a very promising strategy to “bridge” older patients with co-morbidities to the potential curative treatment of allogeneic hematopoietic cell transplantation. In this article, we discuss the role of DNA methyltransferase inhibitors in AML.

  12. The rate of spontaneous mutations in human myeloid cells

    Energy Technology Data Exchange (ETDEWEB)

    Araten, David J., E-mail: david.araten@nyumc.org [Division of Hematology, Department of Veterans Affairs New York Harbor Healthcare System (United States); Division of Hematology, Department of Medicine, NYU School of Medicine and the NYU Langone Cancer Center (United States); Krejci, Ondrej [Division of Experimental Hematology and Cancer Biology, Cincinnati Children' s Hospital Medical Center, Cincinnati, OH (United States); DiTata, Kimberly [Division of Hematology, Department of Medicine, NYU School of Medicine and the NYU Langone Cancer Center (United States); Wunderlich, Mark [Division of Experimental Hematology and Cancer Biology, Cincinnati Children' s Hospital Medical Center, Cincinnati, OH (United States); Sanders, Katie J.; Zamechek, Leah [Division of Hematology, Department of Medicine, NYU School of Medicine and the NYU Langone Cancer Center (United States); Mulloy, James C. [Division of Experimental Hematology and Cancer Biology, Cincinnati Children' s Hospital Medical Center, Cincinnati, OH (United States)

    2013-09-15

    Highlights: • We provide the first measurement of the mutation rate (μ) in human myeloid cells. • μ is measured to be 3.6–23 × 10{sup −7} per cell division. • The AML-ETO and MLL-AF9 fusions do not seem to increase μ. • Cooperating mutations in NRAS, FLT3 and p53 not seem to increase μ. • Hypermutability may be required to explain leukemogenesis. - Abstract: The mutation rate (μ) is likely to be a key parameter in leukemogenesis, but historically, it has been difficult to measure in humans. The PIG-A gene has some advantages for the detection of spontaneous mutations because it is X-linked, and therefore only one mutation is required to disrupt its function. Furthermore, the PIG-A-null phenotype is readily detected by flow cytometry. Using PIG-A, we have now provided the first in vitro measurement of μ in myeloid cells, using cultures of CD34+ cells that are transduced with either the AML-ETO or the MLL-AF9 fusion genes and expanded with cytokines. For the AML-ETO cultures, the median μ value was ∼9.4 × 10{sup −7} (range ∼3.6–23 × 10{sup −7}) per cell division. In contrast, few spontaneous mutations were observed in the MLL-AF9 cultures. Knockdown of p53 or introduction of mutant NRAS or FLT3 alleles did not have much of an effect on μ. Based on these data, we provide a model to predict whether hypermutability must occur in the process of leukemogenesis.

  13. SphK1 inhibitor II (SKI-II) inhibits acute myelogenous leukemia cell growth in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Li; Weng, Wei; Sun, Zhi-Xin; Fu, Xian-Jie; Ma, Jun, E-mail: majuntongrensh1@126.com; Zhuang, Wen-Fang, E-mail: wenfangzhuangmd@163.com

    2015-05-15

    Previous studies have identified sphingosine kinase 1 (SphK1) as a potential drug target for treatment of acute myeloid leukemia (AML). In the current study, we investigated the potential anti-leukemic activity of a novel and specific SphK1 inhibitor, SKI-II. We demonstrated that SKI-II inhibited growth and survival of human AML cell lines (HL-60 and U937 cells). SKI-II was more efficient than two known SphK1 inhibitors SK1-I and FTY720 in inhibiting AML cells. Meanwhile, it induced dramatic apoptosis in above AML cells, and the cytotoxicity by SKI-II was almost reversed by the general caspase inhibitor z-VAD-fmk. SKI-II treatment inhibited SphK1 activation, and concomitantly increased level of sphingosine-1-phosphate (S1P) precursor ceramide in AML cells. Conversely, exogenously-added S1P protected against SKI-II-induced cytotoxicity, while cell permeable short-chain ceramide (C6) aggravated SKI-II's lethality against AML cells. Notably, SKI-II induced potent apoptotic death in primary human AML cells, but was generally safe to the human peripheral blood mononuclear cells (PBMCs) isolated from healthy donors. In vivo, SKI-II administration suppressed growth of U937 leukemic xenograft tumors in severe combined immunodeficient (SCID) mice. These results suggest that SKI-II might be further investigated as a promising anti-AML agent. - Highlights: • SKI-II inhibits proliferation and survival of primary and transformed AML cells. • SKI-II induces apoptotic death of AML cells, but is safe to normal PBMCs. • SKI-II is more efficient than two known SphK1 inhibitors in inhibiting AML cells. • SKI-II inhibits SphK1 activity, while increasing ceramide production in AML cells. • SKI-II dose-dependently inhibits U937 xenograft growth in SCID mice.

  14. SIMILARITIES OF ELDERLY AND THERAPY-RELATED AML

    Directory of Open Access Journals (Sweden)

    Francesco D'Alò

    2011-11-01

    Full Text Available Acute myeloid leukemia (AML is a clonal disorder of the hematopoietic stem cell, typical of the elderly, with a median age of over 60 years at diagnosis. In AML, older age is one of the strongest independent adverse prognostic factor, associated with decreased complete response rate, worse disease-free and overall survival, with highest rates of treatment related mortality, resistant disease and relapse, compared to younger patients. While clinical risk factors do not significantly differ between older and younger patients, outcomes are compromised in elderly patients not only by increased comorbidities and susceptibility to toxicity from therapy, but it is now recognized that elderly AML represents a biologically distinct disease, that is itself more aggressive and less responsive to therapy. In elderly individuals prolonged exposure to environmental carcinogens may be the basis for the aggressive biology of the disease. This may also be the basis for similarities between elderly AML and therapy-related myeloid malignancies, mimicking toxic effects of previous cytotoxic treatments on hematopoietic stem cells. Age is itself a risk factor for t-MN, which are more frequent in elderly patients, where also a shorter latency between treatment of primary tumor and t-MN has been reported. Similarities between therapy-related malignancies and elderly AML include morphological aspects, as the presence of multilineage dysplasia preceding and/or concomitant to the development of leukemia, and adverse cytogenetics, including poor karyotype and chromosome 5 and/or 7 abnormalities. Looking at molecular prognosticators in elderly AML, similar to t-MN,  reduced frequency of favorable factors, as reduced number of NPM1 and CEBPA mutated cases has been observed, together with increased incidence of negative factors, as increased MDR1 expression, accelerated telomere shortening  and frequency of methylation changes. Given the unfavorable prognosis of elderly and

  15. Epigenetic landscape of the TERT promoter: a potential biomarker for high risk AML/MDS.

    Science.gov (United States)

    Zhao, Xin; Tian, Xin; Kajigaya, Sachiko; Cantilena, Caroline R; Strickland, Stephen; Savani, Bipin N; Mohan, Sanjay; Feng, Xingmin; Keyvanfar, Keyvan; Dunavin, Neil; Townsley, Danielle M; Dumitriu, Bogdan; Battiwalla, Minoo; Rezvani, Katayoun; Young, Neal S; Barrett, A John; Ito, Sawa

    2016-11-01

    Although recent observations implicate the importance of telomerase activity in acute myeloid leukaemia (AML), the roles of epigenetic regulations of the TERT gene in leukaemogenesis, drug resistance and clinical prognosis in AML are not fully understood. We developed a quantitative pyrosequencing-based methylation assay covering the TERT proximal promoter and a partial exon 1 (TERTpro/Ex1) region and tested both cell lines and primary leukaemia cells derived from AML and AML with preceding myelodysplastic syndrome (AML/MDS) patients (n = 43). Prognostic impact of methylation status of the upstream TERT promoter region was assessed by the Kaplan-Meier method. The activity of the telomerase inhibitor, imetelstat, was measured using leukaemia cell lines. The TERTpro/Ex1 region was highly methylated in all cell lines and primary leukaemia cells showed diverse methylation profiles. Most cases showed hypermethylated regions at the upstream TERTpro/Ex1 region, which were associated with inferior patient survival. TERTpro/Ex1 methylation status was correlated with the cytotoxicity to imetelstat and its combination with hypomethylating agent enhanced the cytotoxicity of imetelstat. AML cell lines and primary blasts harbour distinct TERTpro/Ex1 methylation profiles that could serve as a prognostic biomarker of AML. However, validation in a large cohort of patients is necessary to confirm our findings.

  16. EVI1 is critical for the pathogenesis of a subset of MLL-AF9–rearranged AMLs

    Science.gov (United States)

    Bindels, Eric M. J.; Havermans, Marije; Lugthart, Sanne; Erpelinck, Claudia; Wocjtowicz, Elizabeth; Krivtsov, Andrei V.; Rombouts, Elwin; Armstrong, Scott A.; Taskesen, Erdogan; Haanstra, Jurgen R.; Beverloo, H. Berna; Döhner, Hartmut; Hudson, Wendy A.; Kersey, John H.; Kumar, Ashish R.

    2012-01-01

    The proto-oncogene EVI1 (ecotropic viral integration site-1), located on chromosome band 3q26, is aberrantly expressed in human acute myeloid leukemia (AML) with 3q26 rearrangements. In the current study, we showed, in a large AML cohort carrying 11q23 translocations, that ∼ 43% of all mixed lineage leukemia (MLL)–rearranged leukemias are EVI1pos. High EVI1 expression occurs in AMLs expressing the MLL-AF6, -AF9, -AF10, -ENL, or -ELL fusion genes. In addition, we present evidence that EVI1pos MLL-rearranged AMLs differ molecularly, morphologically, and immunophenotypically from EVI1neg MLL-rearranged leukemias. In mouse bone marrow cells transduced with MLL-AF9, we show that MLL-AF9 fusion protein maintains Evi1 expression on transformation of Evi1pos HSCs. MLL-AF9 does not activate Evi1 expression in MLL-AF9–transformed granulocyte macrophage progenitors (GMPs) that were initially Evi1neg. Moreover, shRNA-mediated knockdown of Evi1 in an Evi1pos MLL-AF9 mouse model inhibits leukemia growth both in vitro and in vivo, suggesting that Evi1 provides a growth-promoting signal. Using the Evi1pos MLL-AF9 mouse leukemia model, we demonstrate increased sensitivity to chemotherapeutic agents on reduction of Evi1 expression. We conclude that EVI1 is a critical player in tumor growth in a subset of MLL-rearranged AMLs. PMID:22553314

  17. The Proteasome Inhibitor Bortezomib Sensitizes AML with Myelomonocytic Differentiation to TRAIL Mediated Apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Dijk, Marianne van; Murphy, Eoin [Apoptosis Research Center, National University of Ireland, University Road, Galway (Ireland); School of Natural Sciences, National University of Ireland, University Road, Galway (Ireland); Morrell, Ruth [Apoptosis Research Center, National University of Ireland, University Road, Galway (Ireland); School of Natural Sciences, National University of Ireland, University Road, Galway (Ireland); School of Medicine, National University of Ireland, University Road, Galway (Ireland); Knapper, Steven [Department of Haematology, School of Medicine, Cardiff University, Heath Park, CF14 4XN Cardiff (United Kingdom); O' Dwyer, Michael [Apoptosis Research Center, National University of Ireland, University Road, Galway (Ireland); School of Medicine, National University of Ireland, University Road, Galway (Ireland); Samali, Afshin; Szegezdi, Eva, E-mail: eva.szegezdi@nuigalway.ie [Apoptosis Research Center, National University of Ireland, University Road, Galway (Ireland); School of Natural Sciences, National University of Ireland, University Road, Galway (Ireland)

    2011-03-15

    Acute myeloid leukemia (AML) is an aggressive stem cell malignancy that is difficult to treat. There are limitations to the current treatment regimes especially after disease relapse, and therefore new therapeutic agents are urgently required which can overcome drug resistance whilst avoiding unnecessary toxicity. Among newer targeted agents, both tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and proteasome inhibitors show particular promise. In this report we show that a combination of the proteasome inhibitor bortezomib and TRAIL is effective against AML cell lines, in particular, AML cell lines displaying myelomonocytic/monocytic phenotype (M4/M5 AML based on FAB classification), which account for 20-30% of AML cases. We show that the underlying mechanism of sensitization is at least in part due to bortezomib mediated downregulation of c-FLIP and XIAP, which is likely to be regulated by NF-κB. Blockage of NF-κB activation with BMS-345541 equally sensitized myelomonocytic AML cell lines and primary AML blasts to TRAIL.

  18. The Proteasome Inhibitor Bortezomib Sensitizes AML with Myelomonocytic Differentiation to TRAIL Mediated Apoptosis

    Directory of Open Access Journals (Sweden)

    Eva Szegezdi

    2011-03-01

    Full Text Available Acute myeloid leukemia (AML is an aggressive stem cell malignancy that is difficult to treat. There are limitations to the current treatment regimes especially after disease relapse, and therefore new therapeutic agents are urgently required which can overcome drug resistance whilst avoiding unnecessary toxicity. Among newer targeted agents, both tumor necrosis factor (TNF-related apoptosis-inducing ligand (TRAIL and proteasome inhibitors show particular promise. In this report we show that a combination of the proteasome inhibitor bortezomib and TRAIL is effective against AML cell lines, in particular, AML cell lines displaying myelomonocytic/monocytic phenotype (M4/M5 AML based on FAB classification, which account for 20-30% of AML cases. We show that the underlying mechanism of sensitization is at least in part due to bortezomib mediated downregulation of c-FLIP and XIAP, which is likely to be regulated by NF-κB. Blockage of NF-κB activation with BMS-345541 equally sensitized myelomonocytic AML cell lines and primary AML blasts to TRAIL.

  19. In vivo expansion of co-transplanted T cells impacts on tumor re-initiating activity of human acute myeloid leukemia in NSG mice.

    Directory of Open Access Journals (Sweden)

    Malte von Bonin

    Full Text Available Human cells from acute myeloid leukemia (AML patients are frequently transplanted into immune-compromised mouse strains to provide an in vivo environment for studies on the biology of the disease. Since frequencies of leukemia re-initiating cells are low and a unique cell surface phenotype that includes all tumor re-initiating activity remains unknown, the underlying mechanisms leading to limitations in the xenotransplantation assay need to be understood and overcome to obtain robust engraftment of AML-containing samples. We report here that in the NSG xenotransplantation assay, the large majority of mononucleated cells from patients with AML fail to establish a reproducible myeloid engraftment despite high donor chimerism. Instead, donor-derived cells mainly consist of polyclonal disease-unrelated expanded co-transplanted human T lymphocytes that induce xenogeneic graft versus host disease and mask the engraftment of human AML in mice. Engraftment of mainly myeloid cell types can be enforced by the prevention of T cell expansion through the depletion of lymphocytes from the graft prior transplantation.

  20. SphK1 inhibitor II (SKI-II) inhibits acute myelogenous leukemia cell growth in vitro and in vivo.

    Science.gov (United States)

    Yang, Li; Weng, Wei; Sun, Zhi-Xin; Fu, Xian-Jie; Ma, Jun; Zhuang, Wen-Fang

    2015-05-15

    Previous studies have identified sphingosine kinase 1 (SphK1) as a potential drug target for treatment of acute myeloid leukemia (AML). In the current study, we investigated the potential anti-leukemic activity of a novel and specific SphK1 inhibitor, SKI-II. We demonstrated that SKI-II inhibited growth and survival of human AML cell lines (HL-60 and U937 cells). SKI-II was more efficient than two known SphK1 inhibitors SK1-I and FTY720 in inhibiting AML cells. Meanwhile, it induced dramatic apoptosis in above AML cells, and the cytotoxicity by SKI-II was almost reversed by the general caspase inhibitor z-VAD-fmk. SKI-II treatment inhibited SphK1 activation, and concomitantly increased level of sphingosine-1-phosphate (S1P) precursor ceramide in AML cells. Conversely, exogenously-added S1P protected against SKI-II-induced cytotoxicity, while cell permeable short-chain ceramide (C6) aggravated SKI-II's lethality against AML cells. Notably, SKI-II induced potent apoptotic death in primary human AML cells, but was generally safe to the human peripheral blood mononuclear cells (PBMCs) isolated from healthy donors. In vivo, SKI-II administration suppressed growth of U937 leukemic xenograft tumors in severe combined immunodeficient (SCID) mice. These results suggest that SKI-II might be further investigated as a promising anti-AML agent.

  1. CRISPR/Cas9-Directed Reassignment of the GATA1 Initiation Codon in K562 Cells to Recapitulate AML in Down Syndrome

    Directory of Open Access Journals (Sweden)

    Kevin M. Bloh

    2017-06-01

    Full Text Available Using a CRISPR/Cas9 system, we have reengineered a translational start site in the GATA1 gene in K562 cells. This mutation accounts largely for the onset of myeloid leukemia in Down syndrome (ML-DS. For this reengineering, we utilized CRISPR/Cas9 to generate mammalian cell lines that express truncated versions of the Gata1s protein similar to that seen in ML-DS, as determined by analyzing specific genetic alterations resulting from CRISPR/Cas9 cleavage. During this work, 73 cell lines were clonally expanded, with allelic variance analyzed. Using Tracking of Indels by DEcomposition (TIDE and Sanger sequencing, we defined the DNA sequence and variations within each allele. We found significant heterogeneity between alleles in the same clonally expanded cell, as well as among alleles from other clonal expansions. Our data demonstrate and highlight the importance of the randomness of resection promoted by non-homologous end joining after CRISPR/Cas9 cleavage in cells undergoing genetic reengineering. Such heterogeneity must be fully characterized to predict altered functionality inside target tissues and to accurately interpret the associated phenotype. Our data suggest that in cases where the objective is to rearrange specific nucleotides to redirect gene expression in human cells, it is imperative to analyze genetic composition at the individual allelic level.

  2. The CXCR4 inhibitor BL-8040 induces the apoptosis of AML blasts by downregulating ERK, BCL-2, MCL-1 and cyclin-D1 via altered miR-15a/16-1 expression.

    Science.gov (United States)

    Abraham, M; Klein, S; Bulvik, B; Wald, H; Weiss, I D; Olam, D; Weiss, L; Beider, K; Eizenberg, O; Wald, O; Galun, E; Avigdor, A; Benjamini, O; Nagler, A; Pereg, Y; Tavor, S; Peled, A

    2017-04-07

    CXCR4 is a key player in the retention and survival of human acute myeloid leukemia (AML) blasts in the bone marrow (BM) microenvironment. We studied the effects of the CXCR4 antagonist BL-8040 on the survival of AML blasts, and investigated the molecular mechanisms by which CXCR4 signaling inhibition leads to leukemic cell death. Treatment with BL-8040 induced the robust mobilization of AML blasts from the BM. In addition, AML cells exposed to BL-8040 underwent differentiation. Furthermore, BL-8040 induced the apoptosis of AML cells in vitro and in vivo. This apoptosis was mediated by the upregulation of miR-15a/miR-16-1, resulting in downregulation of the target genes BCL-2, MCL-1 and cyclin-D1. Overexpression of miR-15a/miR-16-1 directly induced leukemic cell death. BL-8040-induced apoptosis was also mediated by the inhibition of survival signals via the AKT/ERK pathways. Importantly, treatment with a BCL-2 inhibitor induced apoptosis and act together with BL-8040 to enhance cell death. BL-8040 also synergized with FLT3 inhibitors to induce AML cell death. Importantly, this combined treatment prolonged the survival of tumor-bearing mice and reduced minimal residual disease in vivo. Our results provide a rationale to test combination therapies employing BL-8040 and BCL-2 or FLT3 inhibitors to achieve increased efficacy of these agents.Leukemia advance online publication, 7 April 2017; doi:10.1038/leu.2017.82.

  3. A Polyethylenimine-Containing and Transferrin-Conjugated Lipid Nanoparticle System for Antisense Oligonucleotide Delivery to AML

    Directory of Open Access Journals (Sweden)

    Yiming Yuan

    2016-01-01

    Full Text Available Limited success of antisense oligonucleotides (ASO in clinical anticancer therapy calls for more effective delivery carriers. The goal of this study was to develop a nanoparticle system for delivery of ASO G3139, which targets mRNA of antiapoptotic protein Bcl-2, to acute myeloid leukemia (AML cells. The synthesized nanoparticle Tf-LPN-G3139 contained a small molecular weight polyethylenimine and two cationic lipids as condensing agents, with transferrin on its surface for selective binding and enhanced cellular uptake. The optimized nitrogen to phosphate (N/P ratio was 4 to achieve small particle size and high G3139 entrapment efficiency. The Tf-LPN-G3139 exhibited excellent colloidal stability during storage for at least 12 weeks and remained intact for 4 hours in nuclease-containing serum. The cellular uptake results showed extensive internalization of fluorescence-labelled G3139 in MV4-11 cells through Tf-LPN. Following transfection, Tf-LPN-G3139 at 1 µM ASO level induced 54% Bcl-2 downregulation and >20-fold apoptosis compared to no treatment. When evaluated in mice bearing human xenograft AML tumors, Tf-LPN-G3139 suppressed tumor growth by ~60% at the end of treatment period, accompanied by remarkable pharmacological effect of Bcl-2 inhibition in tumor. In conclusion, Tf-LPN-G3139 is a promising nanoparticle system for ASO G3139 delivery to AML and warrants further investigations.

  4. Two splice-factor mutant leukemia subgroups uncovered at the boundaries of MDS and AML using combined gene expression and DNA-methylation profiling.

    Science.gov (United States)

    Taskesen, Erdogan; Havermans, Marije; van Lom, Kirsten; Sanders, Mathijs A; van Norden, Yvette; Bindels, Eric; Hoogenboezem, Remco; Reinders, Marcel J T; Figueroa, Maria E; Valk, Peter J M; Löwenberg, Bob; Melnick, Ari; Delwel, Ruud

    2014-05-22

    Mutations in splice factor (SF) genes occur more frequently in myelodysplastic syndromes (MDS) than in acute myeloid leukemias (AML). We sequenced complementary DNA from bone marrow of 47 refractory anemia with excess blasts (RAEB) patients, 29 AML cases with low marrow blast cell count, and 325 other AML patients and determined the presence of SF-hotspot mutations in SF3B1, U2AF35, and SRSF2. SF mutations were found in 10 RAEB, 12 AML cases with low marrow blast cell count, and 25 other AML cases. Our study provides evidence that SF-mutant RAEB and SF-mutant AML are clinically, cytologically, and molecularly highly similar. An integrated analysis of genomewide messenger RNA (mRNA) expression profiling and DNA-methylation profiling data revealed 2 unique patient clusters highly enriched for SF-mutant RAEB/AML. The combined genomewide mRNA expression profiling/DNA-methylation profiling signatures revealed 1 SF-mutant patient cluster with an erythroid signature. The other SF-mutant patient cluster was enriched for NRAS/KRAS mutations and showed an inferior survival. We conclude that SF-mutant RAEB/AML constitutes a related disorder overriding the artificial separation between AML and MDS, and that SF-mutant RAEB/AML is composed of 2 molecularly and clinically distinct subgroups. We conclude that SF-mutant disorders should be considered as myeloid malignancies that transcend the boundaries of AML and MDS.

  5. Asparagine synthetase expression is linked with L-asparaginase resistance in TEL-AML1-negative but not TEL-AML1-positive pediatric acute lymphoblastic leukemia.

    Science.gov (United States)

    Stams, Wendy A G; den Boer, Monique L; Holleman, Amy; Appel, Inge M; Beverloo, H Berna; van Wering, Elisabeth R; Janka-Schaub, Gritta E; Evans, William E; Pieters, Rob

    2005-06-01

    Resistance to L-asparaginase in leukemic cells may be caused by an elevated cellular expression of asparagine synthetase (AS). Previously, we reported that high AS expression did not correlate to L-asparaginase resistance in TEL-AML1-positive B-lineage acute lymphoblastic leukemia (ALL). In the present study we confirmed this finding in TEL-AML1-positive patients (n = 28) using microarrays. In contrast, 35 L-asparaginase-resistant TEL-AML1-negative B-lineage ALL patients had a significant 3.5-fold higher AS expression than 43 sensitive patients (P < .001). Using real-time quantitative polymerase chain reaction (RTQ-PCR), this finding was confirmed in an independent group of 39 TEL-AML1-negative B-lineage ALL patients (P = .03). High expression of AS was associated with poor prognosis (4-year probability of disease-free survival [pDFS] 58% +/- 11%) compared with low expression (4-year pDFS 83% +/- 7%; P = .009). We conclude that resistance to l-asparaginase and relapse risk are associated with high expression of AS in TEL-AML1-negative but not TEL-AML1-positive B-lineage ALL.

  6. Using genome-wide CRISPR library screening with library resistant DCK to find new sources of Ara-C drug resistance in AML.

    Science.gov (United States)

    Kurata, Morito; Rathe, Susan K; Bailey, Natashay J; Aumann, Natalie K; Jones, Justine M; Veldhuijzen, G Willemijn; Moriarity, Branden S; Largaespada, David A

    2016-11-03

    Acute myeloid leukemia (AML) can display de novo or acquired resistance to cytosine arabinoside (Ara-C), a primary component of induction chemotherapy. To identify genes capable of independently imposing Ara-C resistance, we applied a genome-wide CRISPR library to human U937 cells and exposed to them to Ara-C. Interestingly, all drug resistant clones contained guide RNAs for DCK. To avoid DCK gene modification, gRNA resistant DCK cDNA was created by the introduction of silent mutations. The CRISPR screening was repeated using the gRNA resistant DCK, and loss of SLC29A was identified as also being capable of conveying Ara-C drug resistance. To determine if loss of Dck results in increased sensitivity to other drugs, we conducted a screen of 446 FDA approved drugs using two Dck-defective BXH-2 derived murine AML cell lines and their Ara-C sensitive parental lines. Both cell lines showed an increase in sensitivity to prednisolone. Guide RNA resistant cDNA rescue was a legitimate strategy and multiple DCK or SLC29A deficient human cell clones were established with one clone becoming prednisolone sensitive. Dck-defective leukemic cells may become prednisolone sensitive indicating prednisolone may be an effective adjuvant therapy in some cases of DCK-negative AML.

  7. Using genome-wide CRISPR library screening with library resistant DCK to find new sources of Ara-C drug resistance in AML

    Science.gov (United States)

    Kurata, Morito; Rathe, Susan K.; Bailey, Natashay J.; Aumann, Natalie K.; Jones, Justine M.; Veldhuijzen, G. Willemijn; Moriarity, Branden S.; Largaespada, David A.

    2016-01-01

    Acute myeloid leukemia (AML) can display de novo or acquired resistance to cytosine arabinoside (Ara-C), a primary component of induction chemotherapy. To identify genes capable of independently imposing Ara-C resistance, we applied a genome-wide CRISPR library to human U937 cells and exposed to them to Ara-C. Interestingly, all drug resistant clones contained guide RNAs for DCK. To avoid DCK gene modification, gRNA resistant DCK cDNA was created by the introduction of silent mutations. The CRISPR screening was repeated using the gRNA resistant DCK, and loss of SLC29A was identified as also being capable of conveying Ara-C drug resistance. To determine if loss of Dck results in increased sensitivity to other drugs, we conducted a screen of 446 FDA approved drugs using two Dck-defective BXH-2 derived murine AML cell lines and their Ara-C sensitive parental lines. Both cell lines showed an increase in sensitivity to prednisolone. Guide RNA resistant cDNA rescue was a legitimate strategy and multiple DCK or SLC29A deficient human cell clones were established with one clone becoming prednisolone sensitive. Dck-defective leukemic cells may become prednisolone sensitive indicating prednisolone may be an effective adjuvant therapy in some cases of DCK-negative AML. PMID:27808171

  8. 地西他滨联合丙戊酸钠对一例AML-M4复发患者原始细胞体外的分化凋亡影响%Synergistic effect of decitabine and valproic acid on differentiation and apoptosis of stem cells of an AML - M4 patient in vitro

    Institute of Scientific and Technical Information of China (English)

    安明和; 王晔恺; 周吉航; 李翊卫; 曾芳

    2012-01-01

    Objective:To investigate the synergistic effect of decitabine(DCA) and valproic acid(VPA) on differentiation and apoptosis of stem cells of an AML - M4 patient in vitro. Methods; The groups were set as follows: control group; DCA alone group A ( 1. 0 μmol/L); DCA alone group B (4. 0 μmol/L); VPA alone group (2.0 nunol/L); combination group A ( DCA 1. 0 μmol/L + VPA 2. 0 mmol/L) ; combination group B ( DCA 4.0 μmol/L + VPA 2.0 mmol/L) . The cells were treated by drug for 48 hours. Then the apoptosis rates, CD117 and CD14 expressions were detected by flow cytometry. Results; Compared with corresponding single drug group,the apoptosis rates and CD14 expressions of the combination group A and combination group B were significantly higher(P <0.01) while CD117 expressions were significantly lower(P< 0.01). Conclusion; Combination of DCA and VPA in vitro can remarkably enhance anti - leukemia effect.%目的:探讨地西他滨( decitabine,DCA)和丙戊酸钠(valproic acid,VPA)联用对AML患者原始细胞体外的影响.方法:设立分组如下:对照组,DCA单药A组(1.0 μmol/L),DCA单药B组(4.0 μmol/L),VPA单药组(2.0 mmol/L),联合用药A组(DCA 1.0 mol/L+VPA 2.0 mmol/L),联合用药B组(DCA4.0 μmol/L+ VPA 2.0 mmol/L),作用48 h.应用流式细胞术检测早期凋亡率和CD117、CD14表达率.结果:相对于各自的单药组,联合用药A组和联合用药B组均能显著提高早期凋亡率和CD14表达,抑制CD117的表达(P<0.01).结论:体外DCA联合VPA能显著加强抗白血病效应.

  9. Human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Abdallah, Basem; Kassem, Moustapha

    2008-01-01

    Mesenchymal stem cells (MSC) are a group of clonogenic cells present among the bone marrow stroma and capable of multilineage differentiation into mesoderm-type cells such as osteoblasts, adipocytes and chondrocytes. Due to their ease of isolation and their differentiation potential, MSC are being...... introduced into clinical medicine in variety of applications and through different ways of administration. Here, we discuss approaches for isolation, characterization and directing differentiation of human mesenchymal stem cells (hMSC). An update of the current clinical use of the cells is also provided....

  10. 干扰核仁磷酸蛋白对人白血病OCI/AML3细胞凋亡的影响及其机制%Effect of nucleophosmin(NPM) 1 knockdown on the apoptosis of OCI/AML3 leukemic cell with mutant NPM1 and its mechanisms

    Institute of Scientific and Technical Information of China (English)

    王娟; 陈莎娜; 全静; 贺金刚; 张帅帅; 鲜敬荣; 张伶

    2014-01-01

    目的:探讨抑制核仁磷酸蛋白(nucleophosmin,NPM)1基因对人白血病细胞凋亡的影响及相关机制.方法:将空载慢病毒pGIPZ和NPM干扰慢病毒shNPM分别感染携带NPM1突变的OCI/AML3白血病细胞株以及对照白血病细胞株HL60.采用qRT-PCR、Western blot和免疫细胞化学法检测白血病细胞的shNPM组以及Mock和pGIPZ组细胞及NPM1A型突变(NPM1-mA)基因和蛋白的表达,流式细胞仪和瑞氏染色法观察OCI/AML3的shNPM组以及Mock和pGIPZ组细胞凋亡情况,qRT-PCR和Western blot检测OCI/AML3的shNPM组以及Mock和pGIPZ组凋亡相关蛋白(Bax/Bcl-2)和p-ERK信号分子表达;流式细胞仪检测丝裂原活化蛋白激酶胞外信号调节激酶信号通路抑制剂(PD98059)处理后OCI/AML3的shNPM组以及Mock和pGIPZ组细胞凋亡率的改变.结果:干扰NPM1明显抑制OCI/AML3细胞株NPM1-mA的mRNA水平(F=20.078;P=0.000,PMock vs shNPM0.001,PpGIPz vs shNPM=0.003,PMock vs pGIPZ=0.333)和蛋白水平,伴有胞质NPM突变蛋白表达明显减弱;干扰NPM1能明显上调OCI/AML3细胞凋亡率(F=25.236,P=0.000,PMock vs shNPM=0.001,PpGIPz vs shNPM=0.001,PMock vs pGIPz=0.729),同时光镜下观察到干扰组细胞出现凋亡小体等凋亡形态学特征.此外,干扰NPM1可上调OCI/AML3细胞中促凋亡分子Bax的蛋白和mRNA表达(F=7.649,P=0.000;PMock vs shNPM=0.015,PpGIPZ vs shNPM=0.015,PMock vs PGIPz=0.984)、下调抗凋亡分子Bcl-2的蛋白和mRNA表达(F=5.190,P=0.000;PMock vs shNPM=0.034,PMock vs shNPM=0.029,PMock vs shNPM=0.909);干扰NPM1能明显下调p-ERK的表达,PD98059抑制剂阻断MAPK通路可促进OCI/AML3细胞凋亡(F=25.643,P=0.007).结论:NPM1突变基因在白血病细胞抗凋亡特性中发挥重要作用,潜在的分子机制可能与ERK信号分子及Bax/Bcl-2表达有关.

  11. DEK oncogene expression during normal hematopoiesis and in Acute Myeloid Leukemia (AML).

    Science.gov (United States)

    Logan, Gemma E; Mor-Vaknin, Nirit; Braunschweig, Till; Jost, Edgar; Schmidt, Pia Verena; Markovitz, David M; Mills, Ken I; Kappes, Ferdinand; Percy, Melanie J

    2015-01-01

    DEK is important in regulating cellular processes including proliferation, differentiation and maintenance of stem cell phenotype. The translocation t(6;9) in Acute Myeloid Leukemia (AML), which fuses DEK with NUP214, confers a poor prognosis and a higher risk of relapse. The over-expression of DEK in AML has been reported, but different studies have shown diminished levels in pediatric and promyelocytic leukemias. This study has characterized DEK expression, in silico, using a large multi-center cohort of leukemic and normal control cases. Overall, DEK was under-expressed in AML compared to normal bone marrow (NBM). Studying specific subtypes of AML confirmed either no significant change or a significant reduction in DEK expression compared to NBM. Importantly, the similarity of DEK expression between AML and NBM was confirmed using immunohistochemistry analysis of tissue mircorarrays. In addition, stratification of AML patients based on median DEK expression levels indicated that DEK showed no effect on the overall survival of patients. DEK expression during normal hematopoiesis did reveal a relationship with specific cell types implicating a distinct function during myeloid differentiation. Whilst DEK may play a potential role in hematopoiesis, it remains to be established whether it is important for leukemagenesis, except when involved in the t(6;9) translocation. Copyright © 2014. Published by Elsevier Inc.

  12. Regulatory T cells-derived IL-35 promotes the growth of adult acute myeloid leukemia blasts.

    Science.gov (United States)

    Tao, Qianshan; Pan, Ying; Wang, Yiping; Wang, Huiping; Xiong, Shudao; Li, Qing; Wang, Jia; Tao, Lili; Wang, Zhitao; Wu, Fan; Zhang, Rui; Zhai, Zhimin

    2015-11-15

    Tumor immune escape mechanism mediated by CD4+CD25+regulatory T cells (Tregs) is a key factor in the pathogenesis of acute myeloid leukemia (AML). IL-35, as a novel inhibitory cytokine, is produced by Tregs specially and regulates functions of Tregs in murine. However, IL-35 expression of Tregs in human is still disputed, and its role in AML is yet to be elucidated. In this study, we found that IL-35 was expressed highly in peripheral blood plasma of adult patients with AML and significantly correlated with the clinical stages of malignancy. Tregs-derived from adult AML patients produced IL-35 in a stimulation-dependent manner. IL-35 promoted AML blasts immune escape by expanding Tregs and inhibiting CD4+CD25-effector T cells (Teffs). Furthermore, IL-35 directly promoted the proliferation of AML blasts and reduced the apoptosis of AML blasts. Together, our study demonstrates that IL-35-derived from Tregs promotes the growth of adult AML blasts, suggesting that IL-35 has an important role in the pathogenesis of AML.

  13. Human TM9SF4 Is a New Gene Down-Regulated by Hypoxia and Involved in Cell Adhesion of Leukemic Cells.

    Directory of Open Access Journals (Sweden)

    Rosa Paolillo

    Full Text Available The transmembrane 9 superfamily protein member 4, TM9SF4, belongs to the TM9SF family of proteins highly conserved through evolution. TM9SF4 homologs, previously identified in many different species, were mainly involved in cellular adhesion, innate immunity and phagocytosis. In human, the function and biological significance of TM9SF4 are currently under investigation. However, TM9SF4 was found overexpressed in human metastatic melanoma and in a small subset of acute myeloid leukemia (AMLs and myelodysplastic syndromes, consistent with an oncogenic function of this gene.In this study, we first analyzed the expression and regulation of TM9SF4 in normal and leukemic cells and identified TM9SF4 as a gene highly expressed in human quiescent CD34+ hematopoietic progenitor cells (HPCs, regulated during monocytic and granulocytic differentiation of HPCs, both lineages giving rise to mature myeloid cells involved in adhesion, phagocytosis and immunity. Then, we found that TM9SF4 is markedly overexpressed in leukemic cells and in AMLs, particularly in M2, M3 and M4 AMLs (i.e., in AMLs characterized by the presence of a more or less differentiated granulocytic progeny, as compared to normal CD34+ HPCs. Proliferation and differentiation of HPCs occurs in hypoxia, a physiological condition in bone marrow, but also a crucial component of cancer microenvironment. Here, we investigated the impact of hypoxia on TM9SF4 expression in leukemic cells and identified TM9SF4 as a direct target of HIF-1α, downregulated in these cells by hypoxia. Then, we found that the hypoxia-mediated downregulation of TM9SF4 expression is associated with a decrease of cell adhesion of leukemic cells to fibronectin, thus demonstrating that human TM9SF4 is a new molecule involved in leukemic cell adhesion.Altogether, our study reports for the first time the expression of TM9SF4 at the level of normal and leukemic hematopoietic cells and its marked expression at the level of AMLs

  14. The role of C/EBPa in PD-1+ CD4+ T cells & modulation of RNR activity prolongs survival of mice with AML

    DEFF Research Database (Denmark)

    Norrie, Ida Christine

    immunosenescence is believed to be changes in T cell distribution, in which the amount of naïve T cells decreases and the amount of memory T cells increases. Cancer cells express immunogenic antigens and mounting of the immune system can essentially recognize and defeat the cancer cells at the initial stages...... PD-1+ CD4+ T cells, suggesting that C/EBPα repress the accumulation of these cells in old mice. Furthermore, C/EBPα-deficiency in the lymphoid compartment had no effect on leukemic development and did not affect the accumulation of PD-1+ CD4+ T cells during development of leukemia. M data indicates...

  15. Coordinate regulation of residual bone marrow function by paracrine trafficking of AML exosomes.

    Science.gov (United States)

    Huan, J; Hornick, N I; Goloviznina, N A; Kamimae-Lanning, A N; David, L L; Wilmarth, P A; Mori, T; Chevillet, J R; Narla, A; Roberts, C T; Loriaux, M M; Chang, B H; Kurre, P

    2015-12-01

    We recently demonstrated that acute myeloid leukemia (AML) cell lines and patient-derived blasts release exosomes that carry RNA and protein; following an in vitro transfer, AML exosomes produce proangiogenic changes in bystander cells. We reasoned that paracrine exosome trafficking may have a broader role in shaping the leukemic niche. In a series of in vitro studies and murine xenografts, we demonstrate that AML exosomes downregulate critical retention factors (Scf, Cxcl12) in stromal cells, leading to hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow. Exosome trafficking also regulates HSPC directly, and we demonstrate declining clonogenicity, loss of CXCR4 and c-Kit expression, and the consistent repression of several hematopoietic transcription factors, including c-Myb, Cebp-β and Hoxa-9. Additional experiments using a model of extramedullary AML or direct intrafemoral injection of purified exosomes reveal that the erosion of HSPC function can occur independent of direct cell-cell contact with leukemia cells. Finally, using a novel multiplex proteomics technique, we identified candidate pathways involved in the direct exosome-mediated modulation of HSPC function. In aggregate, this work suggests that AML exosomes participate in the suppression of residual hematopoietic function that precedes widespread leukemic invasion of the bone marrow directly and indirectly via stromal components.

  16. Hyperdiploidy in Childhood AML Associated with Low Age and AML-M7. A NOPHO-AML Study and Literature Review

    DEFF Research Database (Denmark)

    Sandahl, Julie Damgaard; Abrahamson, Jonas; Heldrup, Jesper

    a literature study searching for hyperdiploid childhood AML (50+) cases using the Mitelman, PubMed, and Scopus databases. The 105 patients from the literature study were compared to the hyperdiploid NOPHO-AML patients. The total cohort of 134 hyperdiploid cases were used to characterize hyperdiploid AML...

  17. Childhood leukemia genetic bottleneck phenomenon related to TEL-AML1: the postulation by a mathematical model

    Institute of Scientific and Technical Information of China (English)

    Petar Ivanovski; Ivan Ivanovski; Dimitrije Nikoli(c); Ivana Jovanovi(c)

    2012-01-01

    Childhood leukemia bottleneck phenomenon is the most mysterious corollary of the prenatal origin discovery of leukemogenic chromosome translocations.The bottleneck is evidence that leukemia initiation,by in utero acquired chromosome translocations that generate functional fusion genes,is far more common than the incidence rate of corresponding leukemia.For childhood TEL-AML1+ acute lymphoblastic leukemia (ALL) this equates to approximately 100 times.Practically this means that among a hundred children born with TEL-AML1 fusion gene,only one child will later in its life develop ALL.The key data necessary for unraveling of this mystery were discovered in 2002.It was the level of TEL-AML1 + cells' frequency.The bottleneck is caused by the very low body TEL-AML1 + cell count.Only one out of a thousand B cells carries TEL-AML1 fusion gene.TEL-AML1+ body cell count is low because TEL-AML1 fusion is generated at cell level of 10a to 10-4 just during the late fetal lymphopoiesis i.e.after the 36th gestational week.

  18. The Aryl Hydrocarbon Receptor Pathway: A Key Component of the microRNA-Mediated AML Signalisome

    Directory of Open Access Journals (Sweden)

    Julia E. Rager

    2012-05-01

    Full Text Available Recent research has spotlighted the role of microRNAs (miRNAs as critical epigenetic regulators of hematopoietic stem cell differentiation and leukemia development. Despite the recent advances in knowledge surrounding epigenetics and leukemia, the mechanisms underlying miRNAs’ influence on leukemia development have yet to be clearly elucidated. Our aim was to identify high ranking biological pathways altered at the gene expression level and under epigenetic control. Specifically, we set out to test the hypothesis that miRNAs dysregulated in acute myeloid leukemia (AML converge on a common pathway that can influence signaling related to hematopoiesis and leukemia development. We identified genes altered in AML patients that are under common regulation of seven key miRNAs. By mapping these genes to a global interaction network, we identified the “AML Signalisome”. The AML Signalisome comprises 53 AML-associated molecules, and is enriched for proteins that play a role in the aryl hydrocarbon receptor (AhR pathway, a major regulator of hematopoiesis. Furthermore, we show biological enrichment for hematopoiesis-related proteins within the AML Signalisome. These findings provide important insight into miRNA-regulated pathways in leukemia, and may help to prioritize targets for disease prevention and treatment.

  19. Cytotoxic T lymphocytes promote cytarabine-induced acute myeloid leukemia cell apoptosis via inhibiting Bcl-2 expression.

    Science.gov (United States)

    Deng, Rui; Fan, Fang-Yi; Yi, Hai; Fu, Li; Zeng, Yan; Wang, Yi; Miao, Xiao-Juan; Shuai, Yan-Rong; He, Guang-Cui; Su, Yi

    2017-08-01

    Acute myeloid leukemia (AML) remains difficult to cure due to its drug tolerance and refractoriness. Immunotherapy is a growing area of cancer research, which has been applied for the treatment of numerous types of cancer, including leukemia. The present study generated AML cell-specific cytotoxic T lymphocytes (CTLs) in vitro and investigated the effect of combining CTL treatment with one of the most commonly used drugs for the treatment of hematological malignancies, cytarabine, on AML cell apoptosis. Firstly, it was observed that monocyte-depleted peripheral blood lymphocytes from healthy donors could be used to generate large numbers of CD3(+)CD8(+) CTLs through immune stimulation. These CD3(+)CD8(+) CTLs could effectively recognize and induce the apoptosis of human Kasumi-3 AML cells. In addition, cytarabine-induced AML cell apoptosis was enhanced by CTL treatment. Western blotting revealed that Bcl-2 expression was downregulated in AML cells following cytarabine and CTL treatment, indicating that the synergistic effect of this treatment on AML cell apoptosis is due to the downregulation of Bcl-2. These results highlight the potential application of CTL immunotherapy for the treatment of AML. Further studies optimizing the specificity and potency of CTLs, and identifying favorable combinations with other chemotherapeutic drug are required.

  20. Childhood leukemia genetic bottleneck phenomenon related to TEL-AML1: the postulation by a mathematical model.

    Science.gov (United States)

    Ivanovski, Petar; Ivanovski, Ivan; Nikolić, Dimitrije; Jovanović, Ivana

    2012-03-01

    Childhood leukemia bottleneck phenomenon is the most mysterious corollary of the prenatal origin discovery of leukemogenic chromosome translocations. The bottleneck is evidence that leukemia initiation, by in utero acquired chromosome translocations that generate functional fusion genes, is far more common than the incidence rate of corresponding leukemia. For childhood TEL-AML1(+) acute lymphoblastic leukemia (ALL) this equates to approximately 100 times. Practically this means that among a hundred children born with TEL-AML1 fusion gene, only one child will later in its life develop ALL. The key data necessary for unraveling of this mystery were discovered in 2002. It was the level of TEL-AML1(+) cells’ frequency. The bottleneck is caused by the very low body TEL-AML1(+) cell count. Only one out of a thousand B cells carries TEL-AML1 fusion gene. TEL-AML1(+) body cell count is low because TEL-AML1 fusion is generated at cell level of 10(-3) to 10(-4) just during the late fetal lymphopoiesis i.e. after the 36th gestational week.

  1. Inhibition of Bcl-2 antiapoptotic members by obatoclax potently enhances sorafenib-induced apoptosis in human myeloid leukemia cells through a Bim-dependent process.

    Science.gov (United States)

    Rahmani, Mohamed; Aust, Mandy Mayo; Attkisson, Elisa; Williams, David C; Ferreira-Gonzalez, Andrea; Grant, Steven

    2012-06-21

    Interactions between the multikinase inhibitor sorafenib and the BH3-mimetic obatoclax (GX15-070) were examined in human acute myeloid leukemia (AML) cells. Treatment with sorafenib/obatoclax induced pronounced apoptosis in and reduced the clonogenic growth of multiple AML lines and primary AML cells but not normal CD34(+) cells. Sorafenib triggered rapid and pronounced Mcl-1 down-regulation accompanied by enhanced binding of Bim to Bcl-2 and Bcl-xL, effects that were abolished by obatoclax coadministration. Notably, shRNA knockdown of Bim, Bak, or Bax, but not Noxa, significantly attenuated obatoclax/sorafenib lethality, whereas ectopic expression of Mcl-1 exerted a protective effect. Furthermore, exposure of leukemia cells to sorafenib and obatoclax markedly induced autophagy, reflected by rapid and pronounced LC3 processing and LC3-green fluorescent protein (GFP) punctate formation. Multiple autophagy inhibitors or VPS34 knockdown, significantly potentiated sorafenib/obatoclax lethality, indicating a cytoprotective role for autophagy in this setting. Finally, studies in a xenograft mouse model revealed that combined sorafenib/obatoclax treatment markedly reduced tumor growth and significantly prolonged survival in association with Mcl-1 down-regulation and apoptosis induction, whereas agents administered individually had only modest effects. These findings suggest that combining sorafenib with agents that inhibit Mcl-1 and Bcl-2/Bcl-xL such as obatoclax may represent a novel and potentially effective strategy in AML.

  2. Response properties of cat AMLS neurons to optic flow stimuli

    Institute of Scientific and Technical Information of China (English)

    LI; Baowang(李宝旺); LI; Bing(李兵); CHEN; Hui(陈辉); XU; Ying(徐颖); DIAO; Yuncheng(刁云程)

    2002-01-01

    Spiral and translation stimuli were used to investigate the response properties of cat AMLS (anteromedial lateral suprasylvian area) neurons to optic flow. The overwhelming majority of cells could be significantly excited by the two modes of stimuli and most responsive cells displayed obvious direction selectivity. It is the first time to find a visual area in mammalian brain preferring rotation stimuli. Two representative hypotheses are discussed here on the neural mechanism of optic flow analysis in visual cortex, and some new viewpoints are proposed to explain the experimental results.

  3. Therapeutic Targeting the Cell Division Cycle 25 (CDC25 Phosphatases in Human Acute Myeloid Leukemia — The Possibility to Target Several Kinases through Inhibition of the Various CDC25 Isoforms

    Directory of Open Access Journals (Sweden)

    Annette K. Brenner

    2014-11-01

    Full Text Available The cell division cycle 25 (CDC25 phosphatases include CDC25A, CDC25B and CDC25C. These three molecules are important regulators of several steps in the cell cycle, including the activation of various cyclin-dependent kinases (CDKs. CDC25s seem to have a role in the development of several human malignancies, including acute myeloid leukemia (AML; and CDC25 inhibition is therefore considered as a possible anticancer strategy. Firstly, upregulation of CDC25A can enhance cell proliferation and the expression seems to be controlled through PI3K-Akt-mTOR signaling, a pathway possibly mediating chemoresistance in human AML. Loss of CDC25A is also important for the cell cycle arrest caused by differentiation induction of malignant hematopoietic cells. Secondly, high CDC25B expression is associated with resistance against the antiproliferative effect of PI3K-Akt-mTOR inhibitors in primary human AML cells, and inhibition of this isoform seems to reduce AML cell line proliferation through effects on NFκB and p300. Finally, CDC25C seems important for the phenotype of AML cells at least for a subset of patients. Many of the identified CDC25 inhibitors show cross-reactivity among the three CDC25 isoforms. Thus, by using such cross-reactive inhibitors it may become possible to inhibit several molecular events in the regulation of cell cycle progression and even cytoplasmic signaling, including activation of several CDKs, through the use of a single drug. Such combined strategies will probably be an advantage in human cancer treatment.

  4. [Effect of 5-aza-2'-deoxycytidine on DAPK gene expression in human HL-60 cells].

    Science.gov (United States)

    Wang, Chun-Yan; Liu, Wen-Jun

    2014-06-01

    This study was aimed to investigate the effect of methylation transferase inhibitor 5-aza-2'-deoxycytidine (5-aza-2dC) of different concentrations on the apoptosis of human acute myeloid leukemia (AML) cell line HL-60 and the expression of DAPK gene in HL-60 cells, as well as to explore the possible anti-AML mechanism of 5-aza-2dC. HL-60 cells were treated by 5-aza-2dC of different concentrations. The effect of 5-aza-2dC on the HL-60 cell morphology was observed by Wright's staining. The effect of 5-aza-2dC on HL-60 cell apoptosis and DAPK mRNA expression was detected by flow cytometry and reverse transcription-polymerize chain reaction (RT-PCR) respectively. The results showed that the 5-aza-2dC induced the apoptosis of HL-60 cells in a concentration-dependent manner; the 5-aza-2dC increased the expression levels of DAPK mRNA in HL-60 cells in a concentration-dependent manner. It is concluded that the apoptosis rate of HL-60 cells and DAPK mRNA expression level displayed a rising trend with 5-aza-2dC concentration increasing. Therefore, DAPK gene may participate in HL-60 cell apoptosis induced by 5-aza-2dC.

  5. Effects of priming with recombinant human granulocyte colony-stimulating factor on conditioning regimen for high-risk acute myeloid leukemia patients undergoing human leukocyte antigen-haploidentical hematopoietic stem cell transplantation: a multicenter randomized controlled study in southwest China.

    Science.gov (United States)

    Gao, Lei; Wen, Qin; Chen, Xinghua; Liu, Yao; Zhang, Cheng; Gao, Li; Kong, Peiyan; Zhang, Yanqi; Li, Yunlong; Liu, Jia; Wang, Qingyu; Su, Yi; Wang, Chunsen; Wang, Sanbin; Zeng, Yun; Sun, Aihua; Du, Xin; Zeng, Dongfeng; Liu, Hong; Peng, Xiangui; Zhang, Xi

    2014-12-01

    HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is an effective and immediate treatment for high-risk acute myeloid leukemia (HR-AML) patients lacking matched donors. Relapse remains the leading cause of death for HR-AML patients after haplo-HSCT. Accordingly, the prevention of relapse remains a challenge in the treatment of HR-AML. In a multicenter randomized controlled trial in southwestern China, 178 HR-AML patients received haplo-HSCT with conditioning regimens involving recombinant human granulocyte colony-stimulating factor (rhG-CSF) or non-rhG-CSF. The cumulative incidences of relapse and graft-versus-host disease (GVHD), 2-year leukemia-free survival (LFS), and overall survival (OS) were evaluated. HR-AML patients who underwent the priming conditioning regimen with rhG-CSF had a lower relapse rate than those who were treated with non-rhG-CSF (38.2%; 95% confidence interval [CI], 28.1% to 48.3% versus 60.7%, 95% CI, 50.5% to 70.8%; P priming group and 31 patients in the non-rhG-CSF-priming group were still alive at the median follow-up time of 42 months (range, 24 to 80 months). The 2-year probabilities of LFS and OS in the rhG-CSF-priming and non-rhG-CSF-priming groups were 55.1% (95% CI, 44.7% to 65.4%) versus 32.6% (95% CI, 22.8% to 42.3%) (P priming group (67.4%; 95% CI, 53.8% to 80.9% versus 41.9%; 95% CI, 27.1% to 56.6%; P priming conditioning regimen is an acceptable choice for HR-AML patients, especially for the patients with no M4/M5/M6 subtype who achieved CR before transplantation.

  6. The role of human equilibrative nucleoside transporter 1 on the cellular transport of the DNA methyltransferase inhibitors 5-azacytidine and CP-4200 in human leukemia cells.

    Science.gov (United States)

    Hummel-Eisenbeiss, Johanna; Hascher, Antje; Hals, Petter-Arnt; Sandvold, Marit Liland; Müller-Tidow, Carsten; Lyko, Frank; Rius, Maria

    2013-09-01

    The nucleoside analog 5-azacytidine is an archetypical drug for epigenetic cancer therapy, and its clinical effectiveness has been demonstrated in the treatment of myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). However, therapy resistance in patients with MDS/AML remains a challenging issue. Membrane proteins that are involved in drug uptake are potential mediators of drug resistance. The responsible proteins for the transport of 5-azacytidine into MDS/AML cells are unknown. We have now systematically analyzed the expression and activity of various nucleoside transporters. We identified the human equilibrative nucleoside transporter 1 (hENT1) as the most abundant nucleoside transporter in leukemia cell lines and in AML patient samples. Transport assays using [¹⁴C]5-azacytidine demonstrated Na⁺-independent uptake of the drug into the cells, which was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBTI), a hENT1 inhibitor. The cellular toxicity of 5-azacytidine and its DNA demethylating activity were strongly reduced after hENT1 inhibition. In contrast, the cellular activity of the 5-azacytidine derivative 5-azacytidine-5'-elaidate (CP-4200), a nucleoside transporter-independent drug, persisted after hENT1 inhibition. A strong dependence of 5-azacytidine-induced DNA demethylation on hENT1 activity was also confirmed by array-based DNA methylation profiling, which uncovered hundreds of loci that became demethylated only when hENT1-mediated transport was active. Our data establish hENT1 as a key transporter for the cellular uptake of 5-azacytidine in leukemia cells and raise the possibility that hENT1 expression might be a useful biomarker to predict the efficiency of 5-azacytidine treatments. Furthermore, our data suggest that CP-4200 may represent a valuable compound for the modulation of transporter-related 5-azacytidine resistances.

  7. Establishment and evaluation of AML-SCID mice model%AML-SCID 小鼠模型的建立及鉴定*

    Institute of Scientific and Technical Information of China (English)

    贾秀红; 张艳君

    2013-01-01

    目的:筛选出高表达HOXA9、HOXA10基因的急性髓系白血病( Acute myeloid leukemia ,AML)细胞株,并建立AML模型,为研究HOXA9、HOXA10与AML的关系奠定基础。方法经RT-PCR检测HOXA9、HOXA10在U937、HL-60、NB4、Jur-kat中的表达情况,筛选出高表达的细胞株;外周血瑞氏染色、骨髓流式细胞仪、脏器HE染色鉴定移植性U937人白血病重度联合免疫缺陷( Severe combined immunodeficiency ,SCID)小鼠模型。结果 HOXA9、HOXA10在U937中均高表达,在HL-60、Jurkat中不表达(或表达水平较低),NB4高表达HOXA10;U937细胞在濒死小鼠骨髓细胞所占的比例为(20.62±9.66%)%,与对照组比较差异有统计学意义( P <0.05);病理检查发现 U937细胞可浸润肝、脾、肺、肾器官。结论 U937高表达HOXA9、HOXA10,是研究HOXA9、HOXA10基因与白血病关系的合适细胞株,并成功建立U937-SCID人M5型急性髓性白血病模型。%Objective To filter out the AML cell line with the HOXA9,HOXA10 gene high expression and establish the AML mod-el,so to lay the foundation for the study of the relationship between HOXA 9,HOXA10,and AML.Methods The expression of HOXA9 and HOXA10 mRNA in U937,HL-60,NB4,Jurkat were detected by reverse transcription PCR .A human leukemia model with severe combined immunodeficiency ( SCID) was intravenously inoculated with U 937 cells.Peripheral blood cells were stained by Wright stain , flow cytometry instrument analyze the ration of CD 45 positive cells in Bone marrow;organs were made into paraffin sections and then stained by Hematoxylin-eosin.Results HOXA9,HOXA10 mRNA level is high in U937 cells,whereas they cannot be detected in HL-60 or Jurkat cells.HOXA10 mRNA level was high in NB4 cells.The proportion of human CD45+cells reached(20.62 ±9.66%)% of bone marrow cells just prior to death of mice ,which was different from the control group obviously ( P﹤0.05 ) .Pathological examina

  8. CHK1 as a therapeutic target to bypass chemoresistance in AML.

    Science.gov (United States)

    David, Laure; Fernandez-Vidal, Anne; Bertoli, Sarah; Grgurevic, Srdana; Lepage, Benoît; Deshaies, Dominique; Prade, Naïs; Cartel, Maëlle; Larrue, Clément; Sarry, Jean-Emmanuel; Delabesse, Eric; Cazaux, Christophe; Didier, Christine; Récher, Christian; Manenti, Stéphane; Hoffmann, Jean-Sébastien

    2016-09-13

    The nucleoside analog cytarabine, an inhibitor of DNA replication fork progression that results in DNA damage, is currently used in the treatment of acute myeloid leukemia (AML). We explored the prognostic value of the expression of 72 genes involved in various aspects of DNA replication in a set of 198 AML patients treated by cytarabine-based chemotherapy. We unveiled that high expression of the DNA replication checkpoint gene CHEK1 is a prognostic marker associated with shorter overall, event-free, and relapse-free survivals and determined that the expression of CHEK1 can predict more frequent and earlier postremission relapse. CHEK1 encodes checkpoint kinase 1 (CHK1), which is activated by the kinase ATR when DNA replication is impaired by DNA damage. High abundance of CHK1 in AML patient cells correlated with higher clonogenic ability and more efficient DNA replication fork progression upon cytarabine treatment. Exposing the patient cells with the high abundance of CHK1 to SCH900776, an inhibitor of the kinase activity of CHK1, reduced clonogenic ability and progression of DNA replication in the presence of cytarabine. These results indicated that some AML cells rely on an efficient CHK1-mediated replication stress response for viability and that therapeutic strategies that inhibit CHK1 could extend current cytarabine-based treatments and overcome drug resistance. Furthermore, monitoring CHEK1 expression could be used both as a predictor of outcome and as a marker to select AML patients for CHK1 inhibitor treatments.

  9. Cellular origin of prognostic chromosomal aberrations in AML patients

    DEFF Research Database (Denmark)

    Mora-Jensen, H.; Jendholm, J.; Rapin, N.

    2015-01-01

    aberrations that were present in the fully transformed committed HPCs together with the prognostic driver aberration. Adding to this vast heterogeneity and complexity of AML genomes and their clonal evolution, a recent study of a murine AML model demonstrated that t(9;11) AML originating from HSCs responded...... poorly to in vivo chemotherapy treatment as compared with t(9;11) AML originating from HPCs....

  10. The detection and classification of blast cell in Leukaemia Acute Promyelocytic Leukaemia (AML M3) blood using simulated annealing and neural networks

    OpenAIRE

    W. Ismail; Hassan, R.; PAYNE, A.; Swift, S

    2011-01-01

    This paper was delivered at AIME 2011: 13th Conference on Artifical Intelligence in Medicine. This paper presents a method for the detection and classification of blast cells in M3 with others sub-types using simulated annealing and neural networks. In this paper, we increased our test result from 10 images to 20 images. We performed Hill Climbing, Simulated Annealing and Genetic Algorithms for detecting the blast cells. As a result, simulated annealing is the “best” heuristic search for d...

  11. GENE EXPRESSION OF NOVEL RETROVIRUS ASSOCIATED WITH HUMAN ACUTE MULOID LEUKEMIA

    Institute of Scientific and Technical Information of China (English)

    许晓华; 徐荣臻; 王世炯; 郑树; 朱宁希; 周旋

    2003-01-01

    Objective: To explore the potentiality of retroviral etiology in human acute myeloid leukemia(AML). Methods: The expression of clone 6#11 in leukemic cell samples from 19 AML cases and peripheral blood mononuclear cells (PBMNCs) from 20 controls was studied by means of Northern blot and reversal transcription polymerase chain reaction (RT-PCR). Results: Northern blot and RT-PCR analyses showed that the expression level of clone 6#11 was significantly higher in AML patients than that in control. Conclusion: Northern blot and RT-PCR analyses revealed that the expression of novel retrovirus were associated with acute myeloid leukemia.

  12. The applicability of the WHO classification in paediatric AML. A NOPHO-AML study

    DEFF Research Database (Denmark)

    Sandahl, Julie D; Kjeldsen, Eigil; Abrahamsson, Jonas;

    2015-01-01

    The World Health Organization (WHO) classification of myeloid leukaemia was revised in 2008. It incorporates newly recognized entities and emphasizes the pivotal role of cytogenetic abnormalities. The aim of this study was to evaluate the usability of the WHO classification when applied to a large......) (44%). In conclusion, the WHO classification allocated 15% to AML-MDS, 44% to NOS and grouped together entities with clearly different outcome, therefore limiting the applicability of the current WHO classification in children with AML....

  13. Cell encoding recombinant human erythropoietin

    Energy Technology Data Exchange (ETDEWEB)

    Beck, A.K.; Withy, R.M.; Zabrecky, J.R.; Masiello, N.C.

    1990-09-04

    This patent describes a C127 cell transformed with a recombinant DNA vector. It comprises: a DNA sequence encoding human erythropoietin, the transformed cell being capable of producing N-linked and O-linked glycosylated human erythropoietin.

  14. Treatment of older patients with AML.

    Science.gov (United States)

    Büchner, Thomas; Berdel, Wolfgang E; Wörmann, Bernhard; Schoch, Claudia; Haferlach, Torsten; Schnittger, Susanne; Kern, Wolfgang; Aul, Carlo; Lengfelder, Eva; Schumacher, Andrea; Reichle, Albrecht; Staib, Peter; Balleisen, Leopold; Eimermacher, Hartmut; Grüneisen, Andreas; Rasche, Herbert; Sauerland, Maria Cristina; Heinecke, Achim; Mesters, Rolf M; Serve, Hubert L; Kienast, Joachim; Hiddemann, Wolfgang

    2005-11-01

    Undertreatment of the older patients with AML can explain, in part, their inferior outcome when compared with that in younger patients. In analogy to the benefit of patients under the age of 60 years from high-dose AraC there are dosage related therapeutic effects in the patients over 60 years in particular for daunorubicin in the induction treatment, and for maintenance versus no maintenance in the post-remission treatment. Utilizing these effects can partly overcome the mostly unfavorable disease biology in older age AML, whereas the role of risk factors involved is not completely understood and the concept of dose-response needs to be requestioned. We recommend an adequate dosage of 60 mg/(m2day) daunorubicin for 3 days in a combination with standard dose AraC and 6-thioguanine given for induction and consolidation and followed by a prolonged monthly maintenance chemotherapy. Further improvements in supportive care may help delivering additional anti-leukemic cytotoxicity. As a novel approach, reduced toxicity preparative regimens may open up allogeneic transplantation for older patients with AML. Other new options like MDR modulators, antibody targeted therapies and tyrosine kinase inhibitors are under clinical investigation. A questionnaire study in patients with AML showed that according to patients' self-assessment intensive and prolonged treatment did not result in decreasing quality of life. This finding did not vary by age under or above 60 years. Given the actual median age in this disease being more than 60 years the adequate management of older age AML remains as the major challenge.

  15. A monoclonal antibody reactive with normal and leukemic human myeloid progenitor cells.

    Science.gov (United States)

    Griffin, J D; Linch, D; Sabbath, K; Larcom, P; Schlossman, S F

    1984-01-01

    Anti-MY9 is an IgG2b murine monoclonal antibody selected for reactivity with immature normal human myeloid cells. The MY9 antigen is expressed by blasts, promyelocytes and myelocytes in the bone marrow, and by monocytes in the peripheral blood. Erythrocytes, lymphocytes and platelets are MY9 negative. All myeloid colony-forming cells (CFU-GM), a fraction of erythroid burst-forming cells (BFU-E) and multipotent progenitors (CFU-GEMM) are MY9 positive. This antigen is further expressed by the leukemic cells of a majority of patients with AML and myeloid CML-BC. Leukemic stem cells (leukemic colony-forming cells, L-CFC) from most patients tested were also MY9 positive. In contrast, MY9 was not detected on lymphocytic leukemias. Anti-MY9 may be a valuable reagent for the purification of hematopoietic colony-forming cells and for the diagnosis of myeloid-lineage leukemias.

  16. 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.

  17. Concurrent targeting Akt and sphingosine kinase 1 by A-674563 in acute myeloid leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Lin [Xiangya Hospital, Central South University, Changsha (China); Shaoyang Central Hospital, Hunan Province (China); Zhang, Yanan; Gao, Meng [The Third Xiangya Hospital, Central South University, Changsha, 410013 (China); Wang, Guangping, E-mail: wangguangping45@sina.com [Xiangya Hospital, Central South University, Changsha (China); Fu, Yunfeng, E-mail: fuyunfeng33163@163.com [The Third Xiangya Hospital, Central South University, Changsha, 410013 (China)

    2016-04-15

    Akt signaling plays a pivotal role in acute myeloid leukemia (AML) development and progression. In the present study, we evaluated the potential anti-AML activity by a novel Akt kinase inhibitor A-674563. Our results showed that A-674563 dose-dependently inhibited survival and proliferation of U937 AML cells and six lines of human AML progenitor cells, yet sparing human peripheral blood mononuclear leukocytes (PBMCs). A-674563 activated caspase-3/9 and apoptosis in the AML cells. Reversely, the pan-caspase inhibitor z-VAD-CHO dramatically alleviated A-674563-induced AML cell apoptosis and cytotoxicity. For the molecular study, we showed that A-674563 blocked Akt activation in U937 cells and human AML progenitor cells. Further, A-674563 decreased sphingosine kinase 1 (SphK1) activity in above AML cells to deplete pro-survival sphingosine-1-phosphate (S1P) and boost pro-apoptotic ceramide production. Such an effect on SphK1 signaling by A-674563 appeared independent of Akt blockage. Significantly, K6PC-5, a novel SphK1 activator, or supplement with S1P attenuated A-674563-induced ceramide production, and subsequent U937 cell death and apoptosis. Importantly, intraperitoneal injection of A-674563 at well-tolerated doses suppressed U937 leukemic xenograft tumor growth in nude mice, whiling significantly improving the animal survival. The results of the current study demonstrate that A-674563 exerts potent anti-leukemic activity in vitro and in vivo, possibly via concurrent targeting Akt and SphK1 signalings. - Highlights: • A-674563 is cytotoxic and anti-proliferative in U937 and AML progenitor cells. • A-674563 activates caspase-3/9 and apoptosis in U937 and AML progenitor cells. • Whiling blocking Akt, A-674563 manipulates other signalings in AML cells. • A-674563 inhibits SphK1 activity in AML cells, independent of Akt blockage. • A-674563 injection inhibits U937 xenograft in vivo growth, and improves mice survival.

  18. [Features of Immunophenotypes and Characteristics of Molecular Biology and Cellular Genetics of AML Patients with CD4 and CD7 Expression].

    Science.gov (United States)

    Liu, Tie-Qiang; Huang, Shan; Yao, Bo; Liu, Zhi-Qing; Yu, Chang-Lin; Qiao, Jian-Hui; Sun, Qi-Yun; Hu, Kai-Xun; Huang, Ya-Jing; Zhang, Rui; Li, Yu-Fang; Bai, Juan; Sun, Yu-Jing; Li, Bing-Xia; Wang, Dong-Mei; Wang, Yi; Guo, Mei

    2016-12-01

    To explore the features of immunophenotypes and the characteristics of molecular biology and cellular genetics of AML patients with CD7 and CD4 expression. The immunophenotypical markers of AML cells were detected by multiple parameter flow cytometry; the expression of WT1, MDK, ETO, PML-RaRa and BCR-ABL were detected by RT-PCR; and cellular features were analyzed by R-band in 304 patients. The patients were divided into three groups according to their immunophenotypes: AML with CD7 expression (CD7 group), AML with CD4 expression(CD4 group) and AML without CD7 and CD4 expression (common AML group). The expression rate and level of HLA-DR in CD7 group were higher than those in the common AML group, and the expression rate of CD33 and CD34 was higher than that in the other two groups. The expression rate and level of CD15, CD64 in the CD4 group were higher than those in the other 2 groups, and the expression rate and level of CD33 were higher than those in the common AML group. WT1 expression in the CD7 group was lower than that in the common AML group. PML-RaRa was not detected in the CD7 group. AML with co-expression of CD4 or CD7 showed more normal karyotype. (15;17) was not found in AML with CD7 expression. AML cells with CD7 expression originate from precursor cells and are blocked in the early phase of hematological development; AML cells with CD4 expression originate from more mature stage of hematological devevelopment and with CD33, CD64 and CD15 high expression; AML cells with CD7 and CD4 expression are characterized by no-specific change of cellular genetics. According to the expression level and intesity of CD4 and CD7, and together with other specific lineage markers, the MRD in AML patients can be quantitatively detected.

  19. Postpartum Diagnosis of AML-M3: A Rare Case Report

    Directory of Open Access Journals (Sweden)

    Manjula Biradar

    2016-07-01

    Full Text Available A 27 year old female gravid 2, para 1, presented at 39 weeks of gestation with pain in abdomen going in for labour, and delivered healthy baby. The patient later had post-partum haemorrhage with Prothrombin Time (PT 10min, platelet count 70,000 cells/cumm. Peripheral smear showed shift to left with myeloblasts, promyelocytes. The diagnosis of Acute Myeloid Leukemia (AML was given and advised for bone marrow examination. The diagnosis AML-M3 was confirmed by marrow study. Unfortunately, patient went in for Disseminated Intravascular Coagulation (DIC and died 2 days after the diagnosis.

  20. Retinoic acid induction of CD38 antigen expression on normal and leukemic human myeloid cells: relationship with cell differentiation.

    Science.gov (United States)

    Prus, Eugenia; Fibach, Eitan

    2003-04-01

    Differentiation in the hematopoietic system involves, among other changes, altered expression of antigens, including the CD34 and CD38 surface antigens. In normal hematopoiesis, the most immature stem cells have the CD34 + CD34 - phenotype. In acute myeloid leukemia (AML), although blasts from most patients are CD38 +, some are CD38 - . AML blasts are blocked at early stages of differentiation; in some leukemic cells this block can be overcome by a variety of agents, including retinoids, that induce maturation into macrophages and granulocytes both in vitro and in vivo. Retinoids can also induce CD38 expression. In the present study, we investigated the relationship between induction of CD38 expression and induction of myeloid differentiation by retinoic acid (RA) in normal and leukemic human hematopoietic cells. In the promyelocytic (PML) CD34 - cell lines, HL60 and CB-1, as well as in normal CD34 + CD34 - hematopietic progenitor cells RA induced both CD38 expression as well as morphological and functional myeloid differentiation that resulted in loss of self-renewal. In contrast, in the myeloblastic CD34 + leukemic cell lines, ML-1 and KG-1a, as well as in primary cultures of cells derived from CD34 + -AML (M0 and M1) patients, RA caused an increase in CD38 + that was not associated with significant differentiation. Yet, long exposure of ML-1, but not KG-1, cells to RA resulted in loss of self-renewal. The results suggest that while in normal hematopoietic cells and in PML CD34 - cells induction of CD38 antigen expression by RA results in terminal differentiation along the myeloid lineage, in early myeloblastic leukemic CD34 + cells, induction of CD38 and differentiation are not functionally related. Since, several lines of evidence suggest that the CD38 - cells are the targets of leukemic transformation, transition of these cellsinto CD38 + phenotype by RA or other drugs may have therapeutic effect, either alone or in conjunction with cytotoxic drugs, regardless

  1. Peptide microarray profiling identifies phospholipase C gamma 1 (PLC-γ1) as a potential target for t(8;21) AML

    Science.gov (United States)

    Mahmud, Hasan; Scherpen, Frank J.G.; de Boer, Tiny Meeuwsen; Lourens, Harm-Jan; Schoenherr, Caroline; Eder, Matthias; Scherr, Michaela; Guryev, Victor; De Bont, Eveline S.

    2017-01-01

    The t(8;21) (q22;q22) chromosomal translocation is one of the most frequent genetic alterations in acute myeloid leukemia (AML) which has a need for improved therapeutic strategies. We found PLC-γ1 as one of the highest phosphorylated peptides in t(8;21) AML samples compared to NBM or CN-AML in our previous peptide microarray. PLC-γ1 is known to play a role in cancer progression, however, the impact of PLC-γ1 in AML is currently unknown. Therefore, we aimed to study the functional role of PLC-γ1 by investigating the cellular growth, survival and its underlying mechanism in t(8;21) AML. In this study, PLC-γ1 expression was significantly higher in t(8;21) AML compared to other karyotypes. The PLC-γ1 protein expression was suppressed in AML1-ETO knock down cells indicating that it might induce kasumi-1 cell death. ShRNA-mediated PLC-γ1 knockdown in kasumi-1 cells significantly blocked cell growth, induced apoptosis and cell cycle arrest which was explained by the increased activation of apoptotic related and cell cycle regulatory protein expressions. Gene expression array analysis showed the up-regulation of apoptotic and DNA damage response genes together with the downregulation of cell growth, proliferation and differentiation genes in the PLC-γ1 suppressed kasumi-1 cells, consistent with the observed phenotypic effects. Importantly, PLC-γ1 suppressed kasumi-1 cells showed higher chemosensitivity to the chemotherapeutic drug treatments and lower cell proliferation upon hypoxic stress. Taken together, these in vitro finding strongly support an important role for PLC-γ1 in the survival of t(8;21) AML mimicking kasumi-1 cells and identify PLC-γ1 as a potential therapeutic target for t(8;21) AML treatment.

  2. Outcome of children with acute myeloid leukaemia (AML) experiencing primary induction failure in the AIEOP AML 2002/01 clinical trial.

    Science.gov (United States)

    Quarello, Paola; Fagioli, Franca; Basso, Giuseppe; Putti, Maria C; Berger, Massimo; Luciani, Matteo; Rizzari, Carmelo; Menna, Giuseppe; Masetti, Riccardo; Locatelli, Franco

    2015-11-01

    Paediatric patients with acute myeloid leukaemia (AML) who fail induction due to primary resistance to chemotherapy account for a significant proportion of cases and have a particularly dismal prognosis. We report the clinical and biological data, and final outcome of 48 paediatric patients with primary-resistant AML enrolled in the Associazione Italiana di Ematologia e Oncologia Pediatrica AML 2002/01 clinical trial. These patients had a significantly higher white blood cell count at diagnosis compared to other AML patients. Cytogenetic and molecular features did not differ between patients with primary induction failure and patients allocated to the high-risk group. For the whole patient population, the probability of overall survival, event-free survival (EFS) and disease-free survival (DFS) was 21·8% ± 6·2, 20·4% ± 5·9, and 49·5% ± 11·3, respectively. Twenty-eight (58%) patients received haematopoietic stem cell transplantation (HSCT); 3 were autologous and 25 were allogeneic. Patients who underwent HSCT had improved EFS (31·2% vs. 5%, P < 0·0001). Only one of the 20 patients who did not receive HSCT is alive and disease free. The 19 patients in complete remission at time of HSCT showed significantly better DFS than the 9 with active disease (46% vs. 0%, P = 0·02). This study represents one of the largest series with long-term follow up of paediatric AML patients with primary refractory disease. Children who underwent transplantation had an encouraging long-term outcome. Disease recurrence remains the major cause of treatment failure; a better understanding of the disease biology is desirable to develop more effective treatment strategies.

  3. Development of Advanced Technologies for Complete Genomic and Proteomic Characterization of Quantized Human Tumor Cells

    Science.gov (United States)

    2015-09-01

    trials: Phase II/III for solid tumors, head & neck, acute myeloid leukemia (AML) Obatoclax BCl-2 inhibitor: induces apoptosis in tumor cells...trials: Phase II/III for AML, myelodysplastic syndromes (MDS), rectal cancer Tanespimycin (17-AAG) Heat shock protein 90 (HSP90) inhibitor: HSP90...immediately after surgical resection, and single cell suspensions are plated in serum-free NeuroCult® NS-A media with B27, epidermal growth factor

  4. Perifosine induces protective autophagy and upregulation of ATG5 in human chronic myelogenous leukemia cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Yin TONG; Yan-yan LIU; Liang-shun YOU; Wen-bin QIAN

    2012-01-01

    Aim:The efficacy of the Akt inhibitor perifosine against chronic myeloid leukemia (CML)cells and its mechanisms of action are unknown.In this study,the cytotoxic effects of perifosine on CML and acute myeloid leukemia (AML)cell lines were compared to elucidate the mechanisms underlying the differences.Methods:Human AML cell lines Kasumi-1 and HL-60,and the CML cell line K562 were used.Cell viability was quantitated using MTT assay.Apoptosis was determined using Annexin V-FITC/propidium iodide and Hoechst staining,which were followed by flow cytometry and fluorescence microscopy analysis,respectively.Caspase pathway activation and the expression of autophagy-related genes were examined using Western blot.Autophagy was studied using electron microscopy,the acridine orange staining method,and GFP-LC3 was examined with fluorescence microscopy.Results:In contrast to AML cell lines,the CML cell lines K562 and K562/G (an imatinib-insensitive CML cell line)were resistant to perifosine (2.5-20 μmol/L)in respect to inhibiting cell growth and inducing apoptosis.Perifosine (2.5,5,and 10 μmol/L)inhibited Akt and its phosphorylation in AML cells,but not in CML cells.Treatment with perifosine (20 μmol/L)resulted in autophagy in CML cells as shown by the increased formation of acidic vesicular organelles and the accumulation of LC3-II.Treatment of CML cells with perifosine (5,10,and 20 μmol/L)dose-dependently upregulated AGT5,but not Beclin 1 at the protein level.Furthermore,inhibition of autophagyby chloroquine (40 nmol/L)significantly suppressed the cell growth and induced apoptosis in CML cells treated with perifosine (20 μmol/L).Conclusion:Our results show that CML cell lines were resistant to the Akt inhibitor perifosine in vitro,which is due to perifosine-induced protective autophagy and upregulation of ATG5.

  5. Ex vivo assays to study self-renewal, long-term expansion, and leukemic transformation of genetically modified human hematopoietic and patient-derived leukemic stem cells.

    Science.gov (United States)

    Sontakke, Pallavi; Carretta, Marco; Capala, Marta; Schepers, Hein; Schuringa, Jan Jacob

    2014-01-01

    With the emergence of the concept of the leukemic stem cell (LSC), assays to study them remain pivotal in understanding (leukemic) stem cell biology. Although the in vivo NOD-SCID or NSG xenotransplantation model is currently still the favored assay of choice in most cases, this system has some limitations as well such as its cost-effectiveness, duration, and lack of engraftability of cells from some acute myeloid leukemia (AML) patients. Here, we describe in vitro assays in which long-term expansion and self-renewal of LSCs isolated from AML patients can be evaluated. We have optimized lentiviral transduction procedures in order to stably express genes of interest or stably downmodulate genes using RNAi in primary AML cells, and these approaches are described in detail here. Also, we describe bone marrow stromal coculture systems in which cobblestone area-forming cell activity, self-renewal, long-term expansion, and in vitro myeloid or lymphoid transformation can be evaluated in human CD34(+) cells of fetal or adult origin that are engineered to express oncogenes. Together, these tools should allow a further molecular elucidation of derailed signal transduction in LSCs.

  6. Fucoidan Suppresses the Growth of Human Acute Promyelocytic Leukemia Cells In Vitro and In Vivo.

    Science.gov (United States)

    Atashrazm, Farzaneh; Lowenthal, Ray M; Woods, Gregory M; Holloway, Adele F; Karpiniec, Samuel S; Dickinson, Joanne L

    2016-03-01

    Fucoidan, a natural component of seaweeds, is reported to have immunomodulatory and anti-tumor effects. The mechanisms underpinning these activities remain poorly understood. In this study, the cytotoxicity and anti-tumor activities of fucoidan were investigated in acute myeloid leukemia (AML) cells. The human AML cell lines NB4, KG1a, HL60, and K562 were treated with fucoidan and cell cycle, cell proliferation, and expression of apoptotic pathways molecules were analyzed. Fucoidan suppressed the proliferation and induced apoptosis through the intrinsic and extrinsic pathways in the acute promyelocytic leukemia (APL) cell lines NB4 and HL60, but not in KG1a and K562 cells. In NB4 cells, apoptosis was caspase-dependent as it was significantly attenuated by pre-treatment with a pan-caspase inhibitor. P21/WAF1/CIP1 was significantly up-regulated leading to cell cycle arrest. Fucoidan decreased the activation of ERK1/2 and down-regulated the activation of AKT through hypo-phosphorylation of Thr(308) residue but not Ser(473). In vivo, a xenograft model using the NB4 cells was employed. Mice were fed with fucoidan and tumor growth was measured following inoculation with NB4 cells. Subsequently, splenic natural killer (NK) cell cytotoxic activity was also examined. Oral doses of fucoidan significantly delayed tumor growth in the xenograft model and increased cytolytic activity of NK cells. Taken together, these data suggest that the selective inhibitory effect of fucoidan on APL cells and its protective effect against APL development in mice warrant further investigation of fucoidan as a useful agent in treatment of certain types of leukemia.

  7. Estudo retrospectivo do tratamento de leucemia mielóide aguda com o transplante de medula óssea: a experiência brasileira Retrospective study of stem cell transplantation for acute myeloid leukemia (AML: the Brazilian experience

    Directory of Open Access Journals (Sweden)

    Nelson Hamerschlak

    2006-03-01

    questionnaires to 16 BMT centers regarding clinical and treatment variables. Statistical analyses concerning autologous BMT (autoBMT and allogeneic BMT (alloBMT were performed using the Kaplan-Meier method and the log-rank test. All p-values were two-tailed. We collected data from 731 patients (205 autoBMT and 526 alloBMT. Median overall survival (OS for autoBMT patients was longer than alloBMT patients (1035 vs. 466 days, p=0.0012. AlloBMT stem cell source (SCS: 73% bone marrow stem cell (BMSC, 23% peripheral blood stem cells (PBSC and 4% umbilical cord blood. Among the autoBMT patients, the SCS was 63% PBSC, 22% BMSC and 15% both. The SCS did not impact on OS. There was no difference in OS between different FAB classifications in the alloBMT group, but in the autoBMT the M3 patients had longer survival. As expected, the main cause of mortality among autoBMT patients was related to disease relapse (60%, while in the alloBMT, to infection (38%. In both groups we found longer OS in first complete remission (1CR compared to second (2CR and other (p<0.0001, and longer OS in de novo AML than in secondary. In the alloBMT group we found more patients with advanced disease (60%, while in the autoBMT group, we found more M3 patients (24%, which could explain the difference in OS. Most of our results are in accordance with IBMTR data. One should consider the fact that this is a retrospective study and our findings should be analysed with caution.

  8. Genome engineering in human cells.

    Science.gov (United States)

    Song, Minjung; Kim, Young-Hoon; Kim, Jin-Soo; Kim, Hyongbum

    2014-01-01

    Genome editing in human cells is of great value in research, medicine, and biotechnology. Programmable nucleases including zinc-finger nucleases, transcription activator-like effector nucleases, and RNA-guided engineered nucleases recognize a specific target sequence and make a double-strand break at that site, which can result in gene disruption, gene insertion, gene correction, or chromosomal rearrangements. The target sequence complexities of these programmable nucleases are higher than 3.2 mega base pairs, the size of the haploid human genome. Here, we briefly introduce the structure of the human genome and the characteristics of each programmable nuclease, and review their applications in human cells including pluripotent stem cells. In addition, we discuss various delivery methods for nucleases, programmable nickases, and enrichment of gene-edited human cells, all of which facilitate efficient and precise genome editing in human cells.

  9. Transmission of clonal chromosomal abnormalities in human hematopoietic stem and progenitor cells surviving radiation exposure

    Energy Technology Data Exchange (ETDEWEB)

    Kraft, Daniela, E-mail: d.kraft@gsi.de [GSI Helmholtz Center for Heavy Ion Research, Department of Biophysics, Planckstr. 1, 64291 Darmstadt (Germany); Institute for Transfusion Medicine und Immunohematology, DRK-Blutspendedienst Baden-Wuerttemberg—Hessen, Johann Wolfgang Goethe-University Hospital, Sandhofstrasse 1, 60528 Frankfurt (Germany); Ritter, Sylvia, E-mail: s.ritter@gsi.de [GSI Helmholtz Center for Heavy Ion Research, Department of Biophysics, Planckstr. 1, 64291 Darmstadt (Germany); Durante, Marco, E-mail: m.durante@gsi.de [GSI Helmholtz Center for Heavy Ion Research, Department of Biophysics, Planckstr. 1, 64291 Darmstadt (Germany); Institute for Condensed Matter Physics, Physics Department, Technical University Darmstadt, Hochschulstraße 6-8, 64289 Darmstadt (Germany); Seifried, Erhard, E-mail: e.seifried@blutspende.de [Institute for Transfusion Medicine und Immunohematology, DRK-Blutspendedienst Baden-Wuerttemberg—Hessen, Johann Wolfgang Goethe-University Hospital, Sandhofstrasse 1, 60528 Frankfurt (Germany); Fournier, Claudia, E-mail: c.fournier@gsi.de [GSI Helmholtz Center for Heavy Ion Research, Department of Biophysics, Planckstr. 1, 64291 Darmstadt (Germany); Tonn, Torsten, E-mail: t.tonn@blutspende.de [Institute for Transfusion Medicine und Immunohematology, DRK-Blutspendedienst Baden-Wuerttemberg—Hessen, Johann Wolfgang Goethe-University Hospital, Sandhofstrasse 1, 60528 Frankfurt (Germany); Technische Universität Dresden, Med. Fakultät Carl Gustav Carus, Institute for Transfusion Medicine Dresden, German Red Cross Blood Donation Service North-East, Blasewitzer Straße 68/70, 01307 Dresden (Germany)

    2015-07-15

    Highlights: • Radiation induced formation and transmission of chromosomal aberrations were assessed. • Cytogenetic analysis was performed in human CD34+ HSPC by mFISH. • We report transmission of stable aberrations in irradiated, clonally expanded HSPC. • Unstable aberrations in clonally expanded HSPC occur independently of irradiation. • Carbon ions and X-rays bear a similar risk for propagation of cytogenetic changes. - Abstract: In radiation-induced acute myeloid leukemia (rAML), clonal chromosomal abnormalities are often observed in bone marrow cells of patients, suggesting that their formation is crucial in the development of the disease. Since rAML is considered to originate from hematopoietic stem and progenitor cells (HSPC), we investigated the frequency and spectrum of radiation-induced chromosomal abnormalities in human CD34{sup +} cells. We then measured stable chromosomal abnormalities, a possible biomarker of leukemia risk, in clonally expanded cell populations which were grown for 14 days in a 3D-matrix (CFU-assay). We compared two radiation qualities used in radiotherapy, sparsely ionizing X-rays and densely ionizing carbon ions (29 and 60–85 keV/μm, doses between 0.5 and 4 Gy). Only a negligible number of de novo arising, unstable aberrations (≤0.05 aberrations/cell, 97% breaks) were measured in the descendants of irradiated HSPC. However, stable aberrations were detected in colonies formed by irradiated HSPC. All cells of the affected colonies exhibited one or more identical aberrations, indicating their clonal origin. The majority of the clonal rearrangements (92%) were simple exchanges such as translocations (77%) and pericentric inversions (15%), which are known to contribute to the development of rAML. Carbon ions were more efficient in inducing cell killing (maximum of ∼30–35% apoptotic cells for 2 Gy carbon ions compared to ∼25% for X-rays) and chromosomal aberrations in the first cell-cycle after exposure (∼70% and

  10. Eriodictyol-induced anti-cancer and apoptotic effects in human hepatocellular carcinoma cells are associated with cell cycle arrest and modulation of apoptosis-related proteins

    Directory of Open Access Journals (Sweden)

    Fang Wang

    2016-06-01

    Full Text Available The objective of the present study was to investigate the anti-cancer effects of eriodictyol in human hepatocellular carcinoma cells (Hep-G2 and normal liver hepatocyte cell line (AML12 along with evaluating its mode of action. Sulforhodamine B assay was used to evaluate the cytotoxic effect of the compound while as fluorescence microscopy was involved to demonstrate the effect of eriodictyol on cellular apoptosis. Flow cytometry was used to investigate the effect of eriodictyol on cell cycle while Western blot analysis revealed the effect on apoptosis-related protein expressions. Results indicate that eriodictyol-induced selective and concentration-dependent cytotoxic effect on Hep-G2 cancer cells while AML12 normal liver cells were very less susceptible to its effect. Eriodictyol-induced apoptosis related morphological changes including chromatin condensation and nuclear fragmentation. It also induced G2/M cell cycle arrest in these cells. Eriodictyol led to up-regulation of Bax and PARP and down-regulation of Bcl-2 protein.

  11. Cellular intrinsic mechanism affecting the outcome of AML treated with Ara-C in a syngeneic mouse model.

    Directory of Open Access Journals (Sweden)

    Wenjun Zhao

    Full Text Available The mechanisms underlying acute myeloid leukemia (AML treatment failure are not clear. Here, we established a mouse model of AML by syngeneic transplantation of BXH-2 derived myeloid leukemic cells and developed an efficacious Ara-C-based regimen for treatment of these mice. We proved that leukemic cell load was correlated with survival. We also demonstrated that the susceptibility of leukemia cells to Ara-C could significantly affect the survival. To examine the molecular alterations in cells with different sensitivity, genome-wide expression of the leukemic cells was profiled, revealing that overall 366 and 212 genes became upregulated or downregulated, respectively, in the resistant cells. Many of these genes are involved in the regulation of cell cycle, cellular proliferation, and apoptosis. Some of them were further validated by quantitative PCR. Interestingly, the Ara-C resistant cells retained the sensitivity to ABT-737, an inhibitor of anti-apoptosis proteins, and treatment with ABT-737 prolonged the life span of mice engrafted with resistant cells. These results suggest that leukemic load and intrinsic cellular resistance can affect the outcome of AML treated with Ara-C. Incorporation of apoptosis inhibitors, such as ABT-737, into traditional cytotoxic regimens merits consideration for the treatment of AML in a subset of patients with resistance to Ara-C. This work provided direct in vivo evidence that leukemic load and intrinsic cellular resistance can affect the outcome of AML treated with Ara-C, suggesting that incorporation of apoptosis inhibitors into traditional cytotoxic regimens merits consideration for the treatment of AML in a subset of patients with resistance to Ara-C.

  12. Nassi-Schneiderman Diagram in HTML Based on AML

    Science.gov (United States)

    Menyhárt, László

    2013-01-01

    In an earlier work I defined an extension of XML called Algorithm Markup Language (AML) for easy and understandable coding in an IDE which supports XML editing (e.g. NetBeans). The AML extension contains annotations and native language (English or Hungarian) tag names used when coding our algorithm. This paper presents a drawing tool with which…

  13. Histological and immunohistochemical features of gingival enlargement in a patient with AML.

    Science.gov (United States)

    Sonoi, Norihiro; Soga, Yoshihiko; Maeda, Hiroshi; Ichimura, Koichi; Yoshino, Tadashi; Aoyama, Kazutoshi; Fujii, Nobuharu; Maeda, Yoshinobu; Tanimoto, Mitsune; Logan, Richard; Raber-Durlacher, Judith; Takashiba, Shogo

    2012-07-01

    Here, we discuss the pathophysiology of leukemia-associated gingival enlargement based on a case of acute myelomonocytic leukemia (AML-M4) with typical gingival enlargement. Uniquely, this patient was well enough to allow full periodontal examination and incisional gingival biopsy to be performed both before and after chemotherapy. The patient was a 39-year-old Japanese woman with AML-M4 showing gingival enlargement. Histological and immunohistochemical features of gingiva and bacterial counts in the periodontal pockets were examined before and after chemotherapy. The results were as follows: (1) infiltration of myelomonocytic blasts in enlarged gingiva; (2) resolution of gingival enlargement with complete remission of AML by anticancer chemotherapy; and (3) the numbers of bacteria in the periodontal pockets were not high and were not altered before or after chemotherapy. In patients with AML-M4, remarkable mucosal enlargement is not generally observed in the body except in the gingiva. We hypothesized that antigens derived from periodontal bacteria, even if they are not present in large numbers, could act as chemoattractants for myelomonocytic leukemic cells.

  14. CXXC5 (Retinoid-Inducible Nuclear Factor, RINF) is a Potential Therapeutic Target in High-Risk Human Acute Myeloid Leukemia

    Science.gov (United States)

    Astori, Audrey; Fredly, Hanne; Aloysius, Thomas Aquinas; Bullinger, Lars; Mas, Véronique Mansat-De; de la Grange, Pierre; Delhommeau, François; Hagen, Karen Marie; Récher, Christian; Dusanter-Fourt, Isabelle; Knappskog, Stian; Lillehaug, Johan Richard

    2013-01-01

    The retinoid-responsive gene CXXC5 localizes to the 5q31.2 chromosomal region and encodes a retinoid-inducible nuclear factor (RINF) that seems important during normal myelopoiesis. We investigated CXXC5/RINF expression in primary human acute myeloid leukemia (AML) cells derived from 594 patients, and a wide variation in CXXC5/RINF mRNA levels was observed both in the immature leukemic myeloblasts and in immature acute lymphoblastic leukemia cells. Furthermore, patients with low-risk cytogenetic abnormalities showed significantly lower levels compared to patients with high-risk abnormalities, and high RINF/CXXC5/ mRNA levels were associated with decreased overall survival for patients receiving intensive chemotherapy for newly diagnosed AML. This association with prognosis was seen both when investigating (i) an unselected patient population as well as for patients with (ii) normal cytogenetic and (iii) core-binding factor AML. CXXC5/RINF knockdown in AML cell lines caused increased susceptibility to chemotherapy-induced apoptosis, and regulation of apoptosis also seemed to differ between primary human AML cells with high and low RINF expression. The association with adverse prognosis together with the antiapoptotic effect of CXXC5/RINF suggests that targeting of CXXC5/RINF should be considered as a possible therapeutic strategy, especially in high-risk patients who show increased expression in AML cells compared with normal hematopoietic cells. PMID:23988457

  15. Cytogenetic findings in adult secondary acute myeloid leukemia (AML): frequency of favorable and adverse chromosomal aberrations do not differ from adult de novo AML

    DEFF Research Database (Denmark)

    Preiss, Birgitte S; Bergman, Olav J; Friis, Lone S;

    2010-01-01

    During a 15-year period, 161 adult patients were diagnosed with secondary acute myeloid leukemia (s-AML) in the region of Southern Denmark. In 73 patients, the AML diagnosis was preceded by myelodysplastic syndrome (MDS-AML), in 31 patients by an antecedent hematologic disease, and in 57 patients...... (P = 0.01). We conclude that population-based cytogenetic studies of adult s-AML and age- and sex-matched de novo AML show comparable distributions of chromosome abnormalities....

  16. Diffusion inside living human cells

    DEFF Research Database (Denmark)

    Leijnse, N.; Jeon, J. -H.; Loft, Steffen

    2012-01-01

    Naturally occurring lipid granules diffuse in the cytoplasm and can be used as tracers to map out the viscoelastic landscape inside living cells. Using optical trapping and single particle tracking we found that lipid granules exhibit anomalous diffusion inside human umbilical vein endothelial...... cells. For these cells the exact diffusional pattern of a particular granule depends on the physiological state of the cell and on the localization of the granule within the cytoplasm. Granules located close to the actin rich periphery of the cell move less than those located towards to the center...... of the cell or within the nucleus. Also, granules in cells which are stressed by intense laser illumination or which have attached to a surface for a long period of time move in a more restricted fashion than those within healthy cells. For granules diffusing in healthy cells, in regions away from the cell...

  17. Autonomous growth potential of leukemia blast cells is associated with poor prognosis in human acute leukemias

    Directory of Open Access Journals (Sweden)

    Jakubowski Ann A

    2009-12-01

    Full Text Available Abstract We have described a severe combined immunodeficiency (SCID mouse model that permits the subcutaneous growth of primary human acute leukemia blast cells into a measurable subcutaneous nodule which may be followed by the development of disseminated disease. Utilizing the SCID mouse model, we examined the growth potential of leukemic blasts from 133 patients with acute leukemia, (67 acute lymphoblastic leukemia (ALL and 66 acute myeloid leukemia (AML in the animals after subcutaneous inoculation without conditioning treatment. The blasts displayed three distinct growth patterns: "aggressive", "indolent", or "no tumor growth". Out of 133 leukemias, 45 (33.8% displayed an aggressive growth pattern, 14 (10.5% displayed an indolent growth pattern and 74 (55.6% did not grow in SCID mice. The growth probability of leukemias from relapsed and/or refractory disease was nearly 3 fold higher than that from patients with newly diagnosed disease. Serial observations found that leukemic blasts from the same individual, which did not initiate tumor growth at initial presentation and/or at early relapse, may engraft and grow in the later stages of disease, suggesting that the ability of leukemia cells for engraftment and proliferation was gradually acquired following the process of leukemia progression. Nine autonomous growing leukemia cell lines were established in vitro. These displayed an aggressive proliferation pattern, suggesting a possible correlation between the capacity of human leukemia cells for autonomous proliferation in vitro and an aggressive growth potential in SCID mice. In addition, we demonstrated that patients whose leukemic blasts displayed an aggressive growth and dissemination pattern in SClD mice had a poor clinical outcome in patients with ALL as well as AML. Patients whose leukemic blasts grew indolently or whose leukemia cells failed to induce growth had a significantly longer DFS and more favorable clinical course.

  18. Silencing AML1-ETO gene expression leads to simultaneous activation of both pro-apoptotic and proliferation signaling.

    Science.gov (United States)

    Spirin, P V; Lebedev, T D; Orlova, N N; Gornostaeva, A S; Prokofjeva, M M; Nikitenko, N A; Dmitriev, S E; Buzdin, A A; Borisov, N M; Aliper, A M; Garazha, A V; Rubtsov, P M; Stocking, C; Prassolov, V S

    2014-11-01

    The t(8;21)(q22;q22) rearrangement represents the most common chromosomal translocation in acute myeloid leukemia (AML). It results in a transcript encoding for the fusion protein AML1-ETO (AE) with transcription factor activity. AE is considered to be an attractive target for treating t(8;21) leukemia. However, AE expression alone is insufficient to cause transformation, and thus the potential of such therapy remains unclear. Several genes are deregulated in AML cells, including KIT that encodes a tyrosine kinase receptor. Here, we show that AML cells transduced with short hairpin RNA vector targeting AE mRNAs have a dramatic decrease in growth rate that is caused by induction of apoptosis and deregulation of the cell cycle. A reduction in KIT mRNA levels was also observed in AE-silenced cells, but silencing KIT expression reduced cell growth but did not induce apoptosis. Transcription profiling of cells that escape cell death revealed activation of a number of signaling pathways involved in cell survival and proliferation. In particular, we find that the extracellular signal-regulated kinase 2 (ERK2; also known as mitogen-activated protein kinase 1 (MAPK1)) protein could mediate activation of 23 out of 29 (79%) of these upregulated pathways and thus may be regarded as the key player in establishing the t(8;21)-positive leukemic cells resistant to AE suppression.

  19. Classification of Acute Myelogenous Leukemia (AML M2 and AML M3) using Momentum Back Propagation from Watershed Distance Transform Segmented Images

    Science.gov (United States)

    Suryani, Esti; Wiharto; Palgunadi, Sarngadi; Nurcahya Pradana, TP

    2017-01-01

    This study uses image processing to analyze white blood cell with leukemia indicated that includes the identification, analysis of shapes and sizes, as well as white blood cell count indicated the symptoms of leukemia. A case study in this research was blood cells, from the type of leukemia Acute Myelogenous Leukemia (AML), M2 and M3 in particular. Image processing operations used for segmentation by utilizing the color conversion from RGB (Red, Green dab Blue) to obtain white blood cell candidates. Furthermore, the white blood cells candidates are separated by other cells with active contour without edge. WBC (White Blood Cell) results still have intersected or overlap condition. Watershed distance transform method can separate overlap of WBC. Furthermore, the separation of the nucleus from the cytoplasm using the HSI (Hue Saturation Intensity). The further characteristic extraction process is done by calculating the area WBC, WBC edge, roundness, the ratio of the nucleus, the mean and standard deviation of pixel intensities. The feature extraction results are used for training and testing in determining the classification of AML: M2 and M3 by using the momentum backpropagation algorithm. The classification process is done by testing the numeric data input from the feature extraction results that have been entered in the database. K-Fold validation is used to divide the amount of training data and to test the classification of AML M2 and M3. The experiment results of eight images trials, the result, was 94.285% per cell accuracy and 75% per image accuracy

  20. Human stromal (mesenchymal) stem cells

    DEFF Research Database (Denmark)

    Aldahmash, Abdullah; Zaher, Walid; Al-Nbaheen, May

    2012-01-01

    Human stromal (mesenchymal) stem cells (hMSC) represent a group of non-hematopoietic stem cells present in the bone marrow stroma and the stroma of other organs including subcutaneous adipose tissue, placenta, and muscles. They exhibit the characteristics of somatic stem cells of self-renewal and......Human stromal (mesenchymal) stem cells (hMSC) represent a group of non-hematopoietic stem cells present in the bone marrow stroma and the stroma of other organs including subcutaneous adipose tissue, placenta, and muscles. They exhibit the characteristics of somatic stem cells of self...... of clinical applications, e.g., non-healing bone fractures and defects and also non-skeletal degenerative diseases like heart failure. Currently, the numbers of clinical trials that employ MSC are increasing. However, several biological and biotechnological challenges need to be overcome to benefit from...

  1. Genomic DNA breakpoints in AML1/RUNX1 and ETO cluster with topoisomerase II DNA cleavage and DNase I hypersensitive sites in t(8;21) leukemia

    Science.gov (United States)

    Zhang, Yanming; Strissel, Pamela; Strick, Reiner; Chen, Jianjun; Nucifora, Giuseppina; Le Beau, Michelle M.; Larson, Richard A.; Rowley, Janet D.

    2002-01-01

    The translocation t(8;21)(q22;q22) is one of the most frequent chromosome translocations in acute myeloid leukemia (AML). AML1/RUNX1 at 21q22 is involved in t(8;21), t(3;21), and t(16;21) in de novo and therapy-related AML and myelodysplastic syndrome as well as in t(12;21) in childhood B cell acute lymphoblastic leukemia. Although DNA breakpoints in AML1 and ETO (at 8q22) cluster in a few introns, the mechanisms of DNA recombination resulting in t(8;21) are unknown. The correlation of specific chromatin structural elements, i.e., topoisomerase II (topo II) DNA cleavage sites, DNase I hypersensitive sites, and scaffold-associated regions, which have been implicated in chromosome recombination with genomic DNA breakpoints in AML1 and ETO in t(8;21) is unknown. The breakpoints in AML1 and ETO were clustered in the Kasumi 1 cell line and in 31 leukemia patients with t(8;21); all except one had de novo AML. Sequencing of the breakpoint junctions revealed no common DNA motif; however, deletions, duplications, microhomologies, and nontemplate DNA were found. Ten in vivo topo II DNA cleavage sites were mapped in AML1, including three in intron 5 and seven in intron 7a, and two were in intron 1b of ETO. All strong topo II sites colocalized with DNase I hypersensitive sites and thus represent open chromatin regions. These sites correlated with genomic DNA breakpoints in both AML1 and ETO, thus implicating them in the de novo 8;21 translocation. PMID:11867721

  2. Significance of murine retroviral mutagenesis for identification of disease genes in human acute myeloid leukemia.

    Science.gov (United States)

    Erkeland, Stefan J; Verhaak, Roel G W; Valk, Peter J M; Delwel, Ruud; Löwenberg, Bob; Touw, Ivo P

    2006-01-15

    Retroviral insertion mutagenesis is considered a powerful tool to identify cancer genes in mice, but its significance for human cancer has remained elusive. Moreover, it has recently been debated whether common virus integrations are always a hallmark of tumor cells and contribute to the oncogenic process. Acute myeloid leukemia (AML) is a heterogeneous disease with a variable response to treatment. Recurrent cytogenetic defects and acquired mutations in regulatory genes are associated with AML subtypes and prognosis. Recently, gene expression profiling (GEP) has been applied to further risk stratify AML. Here, we show that mouse leukemia genes identified by retroviral insertion mutagenesis are more frequently differentially expressed in distinct subclasses of adult and pediatric AML than randomly selected genes or genes located more distantly from a virus integration site. The candidate proto-oncogenes showing discriminative expression in primary AML could be placed in regulatory networks mainly involved in signal transduction and transcriptional control. Our data support the validity of retroviral insertion mutagenesis in mice for human disease and indicate that combining these murine screens for potential proto-oncogenes with GEP in human AML may help to identify critical disease genes and novel pathogenetic networks in leukemia.

  3. The clinical importance of myeloid antigen coexpression and TEL-AML1 mutation in patients with childhood acute lymphoblastic leukemia

    Directory of Open Access Journals (Sweden)

    Ayşen Türedi Yıldırım

    2013-03-01

    Full Text Available Objective: In this study, we aim to investigate the relationship,if any, between clinical features, prognosis, and thecoexpressions and TEL-AML1 mutation in patients withacute lymphoblastic leukemia (ALL.Methods: Eigthy-three patients with acute lymphoblasticleukemia were retrospectively examined. Age, gender,White blood cell count, hemoglobin level, platelet count,ALL subtypei (B or T ALL, risk groups, surface antigensdeteceted by flow cytometry, existence of TEL-AML1 mutations,response, remission and relapse status at 8., 15.ve 33. Days of treatment were recorded and analyzed.Results: 15 (18% out of 83 were identified with aberrantantigen expression. Of these patients, twelve (14.4%had myeloid antigen coexpression (CD13 and/or CD33,two with B cell ALL had CD2 and CD7 coexpressions respectively,one with T cell ALL had CD19 coexpression.No significant differences were found between patientswith and without myeloid antigen coexpression in terms ofhemoglobin levels, white blood cells and platelet counts,responses given on the 8th, 15th, and 30th days on the treatment,risk groups, and relapse (p>0.05. Myeloid antigencoexpression was found in 4 of 13 patients who were identifiedwith TEL-AML1 mutation. No significant relationshipwas found between this mutation and coexpressions. Norelapse and exitus were observed in four patients with coexpressionand TEL-AML1.Conclusion: The prognosis and clinical features showsno statistically significant relationship with the presence ofneither Myeloid antigen expression nor TEL-AML1 mutation.We believe, however, the future studies involving biggersample sizes will prove to be useful in terms of moreconvincing results. J Clin Exp Invest 2013; 4(1: 90-94Key words: Acute lenfoblastic leukemia, coexpression,TEL-AML1 mutation, prognosis

  4. Genome-wide analysis of histone H3 acetylation patterns in AML identifies PRDX2 as an epigenetically silenced tumor suppressor gene

    DEFF Research Database (Denmark)

    Agrawal-Singh, Shuchi; Isken, Fabienne; Agelopoulos, Konstantin

    2012-01-01

    With the use of ChIP on microarray assays in primary leukemia samples, we report that acute myeloid leukemia (AML) blasts exhibit significant alterations in histone H3 acetylation (H3Ac) levels at > 1000 genomic loci compared with CD34+ progenitor cells. Importantly, core promoter regions tended......, which correlated with low mRNA and protein expression. We also observed DNA hypermethylation at the PRDX2 promoter in AML. Low protein expression of the antioxidant PRDX2 gene was clinically associated with poor prognosis in patients with AML. Functionally, PRDX2 acted as inhibitor of myeloid cell...... as an epigenetically silenced growth suppressor, suggesting a possible role of ROS in the malignant phenotype in AML....

  5. Hyperforin inhibits Akt1 kinase activity and promotes caspase-mediated apoptosis involving Bad and Noxa activation in human myeloid tumor cells.

    Directory of Open Access Journals (Sweden)

    Faten Merhi

    Full Text Available BACKGROUND: The natural phloroglucinol hyperforin HF displays anti-inflammatory and anti-tumoral properties of potential pharmacological interest. Acute myeloid leukemia (AML cells abnormally proliferate and escape apoptosis. Herein, the effects and mechanisms of purified HF on AML cell dysfunction were investigated in AML cell lines defining distinct AML subfamilies and primary AML cells cultured ex vivo. METHODOLOGY AND RESULTS: HF inhibited in a time- and concentration-dependent manner the growth of AML cell lines (U937, OCI-AML3, NB4, HL-60 by inducing apoptosis as evidenced by accumulation of sub-G1 population, phosphatidylserine externalization and DNA fragmentation. HF also induced apoptosis in primary AML blasts, whereas normal blood cells were not affected. The apoptotic process in U937 cells was accompanied by downregulation of anti-apoptotic Bcl-2, upregulation of pro-apoptotic Noxa, mitochondrial membrane depolarization, activation of procaspases and cleavage of the caspase substrate PARP-1. The general caspase inhibitor Z-VAD-fmk and the caspase-9- and -3-specific inhibitors, but not caspase-8 inhibitor, significantly attenuated apoptosis. HF-mediated apoptosis was associated with dephosphorylation of active Akt1 (at Ser(473 and Akt1 substrate Bad (at Ser(136 which activates Bad pro-apoptotic function. HF supppressed the kinase activity of Akt1, and combined treatment with the allosteric Akt1 inhibitor Akt-I-VIII significantly enhanced apoptosis of U937 cells. SIGNIFICANCE: Our data provide new evidence that HF's pro-apoptotic effect in AML cells involved inhibition of Akt1 signaling, mitochondria and Bcl-2 members dysfunctions, and activation of procaspases -9/-3. Combined interruption of mitochondrial and Akt1 pathways by HF may have implications for AML treatment.

  6. Human embryonic stem cells handbook

    Directory of Open Access Journals (Sweden)

    Carlo Alberto Redi

    2013-03-01

    Full Text Available After the Nobel prize in physiology or medicine was awarded jointly to Sir John Gurdon and Shinya Yamanaka for the discovery that mature cells can be reprogrammed to become pluripotent it became imperative to write down the review for a book entirely devoted to human embryonic stem cells (hES, those cells that are a urgent need for researchers, those cells that rekindle the ethical debates and finally, last but not least, those cells whose study paved the way to obtain induced pluripotent stem cells by the OSKC’s Yamanaka method (the OSKC acronim refers, for those not familiar with the topic, to the four stemness genes used to transfect somatic fibroblasts: Oct4, Sox2, Klf4 and c-Myc....

  7. Curcumin reduces expression of Bcl-2, leading to apoptosis in daunorubicin-insensitive CD34+ acute myeloid leukemia cell lines and primary sorted CD34+ acute myeloid leukemia cells

    Science.gov (United States)

    2011-01-01

    Background Acute myeloid leukemia (AML) is an immunophenotypically heterogenous malignant disease, in which CD34 positivity is associated with poor prognosis. CD34+ AML cells are 10-15-fold more resistant to daunorubicin (DNR) than CD34- AML cells. Curcumin is a major component of turmeric that has shown cytotoxic activity in multiple cancers; however, its anti-cancer activity has not been well studied in DNR-insensitive CD34+ AML cells. The aim of this study was to therefore to explore curcumin-induced cytotoxicity in DNR-insensitive CD34+ AML cell lines (KG1a, Kasumi-1), DNR-sensitive U937 AML cells, and primary CD34+ AML bone-marrow-derived cells. Methods Primary human CD34+ cells were isolated from peripheral blood mononuclear cells or bone marrow mononuclear cells using a CD34 MicroBead kit. The growth inhibitory effects of curcumin were evaluated by MTT and colony-formation assays. Cell cycle distribution was examined by propidium iodide (PI) assay. Apoptosis was analyzed by Wright-Giemsa, Hoechst 33342 and Annexin-V/PI staining assays. The change in mitochondrial membrane potential (MMP) was examined by JC-1 staining and flow cytometry. Expression of apoptosis-related proteins was determined by reverse transcription-polymerase chain reaction and Western blotting. Short interfering RNA (siRNA) against Bcl-2 was used in CD34+ KG1a and Kasumi-1 cells incubated with/without DNR. Results Curcumin inhibited proliferation and induced apoptosis and G1/S arrest in both DNR-insensitive KG1a, Kasumi-1 and DNR-sensitive U937 cells. Curcumin-induced apoptosis was associated with reduced expression of both Bcl-2 mRNA and protein, subsequent loss of MMP, and activation of caspase-3 followed by PARP degradation. Curcumin synergistically enhanced the cytotoxic effect of DNR in DNR-insensitive KG1a and Kasumi-1 cells, consistent with decreased Bcl-2 expression. Accordingly, siRNA against Bcl-2 increased the susceptibility of KG1a and Kasumi-1 cells to DNR-induced apoptosis

  8. Curcumin reduces expression of Bcl-2, leading to apoptosis in daunorubicin-insensitive CD34+ acute myeloid leukemia cell lines and primary sorted CD34+ acute myeloid leukemia cells

    Directory of Open Access Journals (Sweden)

    Huang Sheng-Shan

    2011-05-01

    Full Text Available Abstract Background Acute myeloid leukemia (AML is an immunophenotypically heterogenous malignant disease, in which CD34 positivity is associated with poor prognosis. CD34+ AML cells are 10-15-fold more resistant to daunorubicin (DNR than CD34- AML cells. Curcumin is a major component of turmeric that has shown cytotoxic activity in multiple cancers; however, its anti-cancer activity has not been well studied in DNR-insensitive CD34+ AML cells. The aim of this study was to therefore to explore curcumin-induced cytotoxicity in DNR-insensitive CD34+ AML cell lines (KG1a, Kasumi-1, DNR-sensitive U937 AML cells, and primary CD34+ AML bone-marrow-derived cells. Methods Primary human CD34+ cells were isolated from peripheral blood mononuclear cells or bone marrow mononuclear cells using a CD34 MicroBead kit. The growth inhibitory effects of curcumin were evaluated by MTT and colony-formation assays. Cell cycle distribution was examined by propidium iodide (PI assay. Apoptosis was analyzed by Wright-Giemsa, Hoechst 33342 and Annexin-V/PI staining assays. The change in mitochondrial membrane potential (MMP was examined by JC-1 staining and flow cytometry. Expression of apoptosis-related proteins was determined by reverse transcription-polymerase chain reaction and Western blotting. Short interfering RNA (siRNA against Bcl-2 was used in CD34+ KG1a and Kasumi-1 cells incubated with/without DNR. Results Curcumin inhibited proliferation and induced apoptosis and G1/S arrest in both DNR-insensitive KG1a, Kasumi-1 and DNR-sensitive U937 cells. Curcumin-induced apoptosis was associated with reduced expression of both Bcl-2 mRNA and protein, subsequent loss of MMP, and activation of caspase-3 followed by PARP degradation. Curcumin synergistically enhanced the cytotoxic effect of DNR in DNR-insensitive KG1a and Kasumi-1 cells, consistent with decreased Bcl-2 expression. Accordingly, siRNA against Bcl-2 increased the susceptibility of KG1a and Kasumi-1 cells to

  9. CNS Involvement in AML Patient Treated with 5-Azacytidine

    Directory of Open Access Journals (Sweden)

    Diamantina Vasilatou

    2014-01-01

    Full Text Available Central nervous system (CNS involvement in acute myeloid leukemia (AML is a rare complication of the disease and is associated with poor prognosis. Sometimes the clinical presentation can be unspecific and the diagnosis can be very challenging. Here we report a case of CNS infiltration in a patient suffering from AML who presented with normal complete blood count and altered mental status.

  10. Epigenetic priming of AML blasts for all-trans retinoic acid-induced differentiation by the HDAC class-I selective inhibitor entinostat.

    Directory of Open Access Journals (Sweden)

    Nadja Blagitko-Dorfs

    Full Text Available All-trans retinoic acid (ATRA has only limited single agent activity in AML without the PML-RARα fusion (non-M3 AML. In search of a sensitizing strategy to overcome this relative ATRA resistance, we investigated the potency of the HDAC class-I selective inhibitor entinostat in AML cell lines Kasumi-1 and HL-60 and primary AML blasts. Entinostat alone induced robust differentiation of both cell lines, which was enhanced by the combination with ATRA. This "priming" effect on ATRA-induced differentiation was at least equivalent to that achieved with the DNA hypomethylating agent decitabine, and could overall be recapitulated in primary AML blasts treated ex vivo. Moreover, entinostat treatment established the activating chromatin marks acH3, acH3K9, acH4 and H3K4me3 at the promoter of the RARβ2 gene, an essential mediator of retinoic acid (RA signaling in different solid tumor models. Similarly, RARβ2 promoter hypermethylation (which in primary blasts from 90 AML/MDS patients was surprisingly infrequent could be partially reversed by decitabine in the two cell lines. Re-induction of the epigenetically silenced RARβ2 gene was achieved only when entinostat or decitabine were given prior to ATRA treatment. Thus in this model, reactivation of RARβ2 was not necessarily required for the differentiation effect, and pharmacological RARβ2 promoter demethylation may be a bystander phenomenon rather than an essential prerequisite for the cellular effects of decitabine when combined with ATRA. In conclusion, as a "priming" agent for non-M3 AML blasts to the differentiation-inducing effects of ATRA, entinostat is at least as active as decitabine, and both act in part independently from RARβ2. Further investigation of this treatment combination in non-M3 AML patients is therefore warranted, independently of RARβ2 gene silencing by DNA methylation.

  11. Patient-individualized CD8⁺ cytolytic T-cell therapy effectively combats minimal residual leukemia in immunodeficient mice.

    Science.gov (United States)

    Distler, Eva; Albrecht, Jana; Brunk, Ariane; Khan, Shamsul; Schnürer, Elke; Frey, Michaela; Mottok, Anja; Jordán-Garrote, Ana-Laura; Brede, Christian; Beilhack, Andreas; Mades, Andreas; Tomsitz, Dirk; Theobald, Matthias; Herr, Wolfgang; Hartwig, Udo F

    2016-03-01

    Adoptive transfer of donor-derived cytolytic T-lymphocytes (CTL) has evolved as a promising strategy to improve graft-versus-leukemia (GvL) effects in allogeneic hematopoietic stem-cell transplantation. However, durable clinical responses are often hampered by limited capability of transferred T cells to establish effective and sustained antitumor immunity in vivo. We therefore analyzed GvL responses of acute myeloid leukemia (AML)-reactive CD8(+) CTL with central and effector memory phenotype in a new allogeneic donor-patient specific humanized mouse model. CTL lines and clones obtained upon stimulation of naive CD45RA(+) donor CD8(+) T cells with either single HLA antigen-mismatched or HLA-matched primary AML blasts, respectively, elicited strong leukemia reactivity during cytokine-optimized short to intermediate (i.e., 2-8 weeks) culture periods. Single doses of CTL were intravenously infused into NOD/scidIL2Rcg(null) mice when engraftment with patient AML reached bone marrow infiltration of 1-5%, clinically defining minimal residual disease status. This treatment resulted in complete regression of HLA-mismatched and strong reduction of HLA-matched AML infiltration, respectively. Most importantly, mice receiving AML-reactive CTL showed significantly prolonged survival. Transferred CTL were detectable in murine bone marrow and spleen and demonstrated sustained AML-reactivity ex vivo. Moreover, injections with human IL-15 clearly promoted CTL persistence. In summary, we show that naive donor-derived CD8(+) CTL effectively combat patient AML blasts in immunodeficient mice. The donor-patient specific humanized mouse model appears suitable to evaluate therapeutic efficacy of AML-reactive CTL before adoptive transfer into patients. It may further help to identify powerful leukemia rejection antigens and T-cell receptors for redirecting immunity to leukemias even in a patient-individualized manner. © 2015 UICC.

  12. Screening for MLL tandem duplication in 387 unselected patients with AML identify a prognostically unfavorable subset of AML.

    Science.gov (United States)

    Schnittger, S; Kinkelin, U; Schoch, C; Heinecke, A; Haase, D; Haferlach, T; Büchner, T; Wörmann, B; Hiddemann, W; Griesinger, F

    2000-05-01

    Partial tandem duplications of the MLL gene have been associated with trisomy 11 in acute myeloid leukemia (AML) and recently, have also been reported for karyotypically normal AML. In order to test the incidence and prognostic importance of this molecular marker, we have analyzed eight cases of AML with trisomy 11 and 387 unselected consecutive cases with AML for partial duplications of the MLL gene. Patients with normal karyotypes and those with various chromosome aberrations were included. De novo as well as secondary leukemias including all FAB subtypes were analyzed. Performing a one-step RT-PCR with 35 cycles using an exon 9 forward primer and an exon 3 reverse primer partial tandem duplications of the MLL gene were demonstrated in 3/8 (37.5%) patients with trisomy 11. In addition, 13/387 (3.4%) of unselected cases revealed a tandem duplication. Ten of these 13 cases were cytogenetically normal, the other three cases had secondary AML after MDS, three were therapy-related AML (t-AML). Of the 16 MLL-duplication positive cases, seven were classified as FAB M2, two as M1, five as M4, one as M0, one as M5b. The mean age was 62.3 years for patients with MLL duplication vs 50.3 years for the control group. Of 15 adult patients, 12 received treatment. Of these, three were nonresponders, five had early relapse (common than previously reported; (2) are preferentially observed in AML with normal karyotypes, but can also be found in the presence of chromosome alterations; (3) are not strongly associated with an FAB subtype; (4) were not observed with the prognostically favorable t(8;21), inv(16), and t(15;17), other recurrent translocations, or in complex karyotypes; and (5) identifies a subgroup of patients with an unfavorable prognosis.

  13. MicroRNA-150 Expression Induces Myeloid Differentiation of Human Acute Leukemia Cells and Normal Hematopoietic Progenitors

    Science.gov (United States)

    Morris, Valerie A.; Zhang, Ailin; Yang, Taimei; Stirewalt, Derek L.; Ramamurthy, Ranjani; Meshinchi, Soheil; Oehler, Vivian G.

    2013-01-01

    In acute myeloid leukemia (AML) and blast crisis (BC) chronic myeloid leukemia (CML) normal differentiation is impaired. Differentiation of immature stem/progenitor cells is critical for normal blood cell function. MicroRNAs (miRNAs or miRs) are small non-coding RNAs that interfere with gene expression by degrading messenger RNAs (mRNAs) or blocking protein translation. Aberrant miRNA expression is a feature of leukemia and miRNAs also play a significant role in normal hematopoiesis and differentiation. We have identified miRNAs differentially expressed in AML and BC CML and identified a new role for miR-150 in myeloid differentiation. Expression of miR-150 is low or absent in BC CML and AML patient samples and cell lines. We have found that expression of miR-150 in AML cell lines, CD34+ progenitor cells from healthy individuals, and primary BC CML and AML patient samples at levels similar to miR-150 expression in normal bone marrow promotes myeloid differentiation of these cells. MYB is a direct target of miR-150, and we have identified that the observed phenotype is partially mediated by MYB. In AML cell lines, differentiation of miR-150 expressing cells occurs independently of retinoic acid receptor α (RARA) signaling. High-throughput gene expression profiling (GEP) studies of the AML cell lines HL60, PL21, and THP-1 suggest that activation of CEPBA, CEBPE, and cytokines associated with myeloid differentiation in miR-150 expressing cells as compared to control cells contributes to myeloid differentiation. These data suggest that miR-150 promotes myeloid differentiation, a previously uncharacterized role for this miRNA, and that absent or low miR-150 expression contributes to blocked myeloid differentiation in acute leukemia cells. PMID:24086639

  14. MicroRNA-150 expression induces myeloid differentiation of human acute leukemia cells and normal hematopoietic progenitors.

    Directory of Open Access Journals (Sweden)

    Valerie A Morris

    Full Text Available In acute myeloid leukemia (AML and blast crisis (BC chronic myeloid leukemia (CML normal differentiation is impaired. Differentiation of immature stem/progenitor cells is critical for normal blood cell function. MicroRNAs (miRNAs or miRs are small non-coding RNAs that interfere with gene expression by degrading messenger RNAs (mRNAs or blocking protein translation. Aberrant miRNA expression is a feature of leukemia and miRNAs also play a significant role in normal hematopoiesis and differentiation. We have identified miRNAs differentially expressed in AML and BC CML and identified a new role for miR-150 in myeloid differentiation. Expression of miR-150 is low or absent in BC CML and AML patient samples and cell lines. We have found that expression of miR-150 in AML cell lines, CD34+ progenitor cells from healthy individuals, and primary BC CML and AML patient samples at levels similar to miR-150 expression in normal bone marrow promotes myeloid differentiation of these cells. MYB is a direct target of miR-150, and we have identified that the observed phenotype is partially mediated by MYB. In AML cell lines, differentiation of miR-150 expressing cells occurs independently of retinoic acid receptor α (RARA signaling. High-throughput gene expression profiling (GEP studies of the AML cell lines HL60, PL21, and THP-1 suggest that activation of CEPBA, CEBPE, and cytokines associated with myeloid differentiation in miR-150 expressing cells as compared to control cells contributes to myeloid differentiation. These data suggest that miR-150 promotes myeloid differentiation, a previously uncharacterized role for this miRNA, and that absent or low miR-150 expression contributes to blocked myeloid differentiation in acute leukemia cells.

  15. The cytokine-mediated crosstalk between primary human acute myeloid cells and mesenchymal stem cells alters the local cytokine network and the global gene expression profile of the mesenchymal cells

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    Håkon Reikvam

    2015-11-01

    Full Text Available Interactions between acute myeloid leukemia (AML blasts and neighboring stromal cells are important for disease development and chemosensitivity. However, the molecular mechanisms involved in the cytokine-mediated crosstalk between mesenchymal stem cells (MSCs and AML cells are largely unknown. Leukemic cells derived from 18 unselected AML patients were cultured with bone marrow MSCs derived from healthy donors; the populations then being separated by a semipermeable membrane. Coculture had only minor effects on MSC proliferation. The unique cytokine network in cocultures was determined by high constitutive MSC release of certain cytokines (especially IL-6 and vascular endothelial growth factor and constitutive release of a wide range of soluble mediators by primary AML cells. However, the AML cell release varied considerably between patients, and these differences between patients were also reflected in the coculture levels even though supra-additive effects were seen for many mediators. These effects on the local cytokine network were dependent on a functional crosstalk between the two cell subsets. The crosstalk altered the global gene expression profile of the MSCs, especially expression of genes encoding proteins involved in downstream signaling from Toll like receptors, NFκB signaling and CCL/CXCL chemokine release. Thus, primary AML cells alter the functional phenotype of normal MSCs.

  16. ERG and FLI1 binding sites demarcate targets for aberrant epigenetic regulation by AML1-ETO in acute myeloid leukemia

    NARCIS (Netherlands)

    Martens, Joost H. A.; Mandoli, Amit; Simmer, Femke; Wierenga, Bart-Jan; Saeed, Sadia; Singh, Abhishek A.; Altucci, Lucia; Vellenga, Edo; Stunnenberg, Hendrik G.

    2012-01-01

    ERG and FLI1 are closely related members of the ETS family of transcription factors and have been identified as essential factors for the function and maintenance of normal hematopoietic stem cells. Here genome-wide analysis revealed that both ERG and FLI1 occupy similar genomic regions as AML1-ETO

  17. Protracted and variable latency of acute lymphoblastic leukemia after TEL-AML1 gene fusion in utero

    NARCIS (Netherlands)

    Wiemels, JL; Ford, AM; Van Wering, ER; Postma, A; Greaves, M

    1999-01-01

    We report a pair of identical twins with concordant acute lymphoblastic leukemia (ALL). Unusually, their diagnoses were spaced 9 years apart at ages 5 and 14, Leukemic cells in both twins had a TEL-AML1 rearrangement, which was characterized at the DNA level by an adaptation of a long distance polym

  18. Overproduction of NOX-derived ROS in AML promotes proliferation and is associated with defective oxidative stress signaling.

    Science.gov (United States)

    Hole, Paul S; Zabkiewicz, Joanna; Munje, Chinmay; Newton, Zarabeth; Pearn, Lorna; White, Paul; Marquez, Nuria; Hills, Robert K; Burnett, Alan K; Tonks, Alex; Darley, Richard L

    2013-11-07

    Excessive production of reactive oxygen species (ROS) is frequently observed in cancer and is known to strongly influence hematopoietic cell function. Here we report that extracellular ROS production is strongly elevated (mean >10-fold) in >60% of acute myeloid leukemia (AML) patients and that this increase is attributable to constitutive activation of nicotinamide adenine dinucleotide phosphate oxidases (NOX). In contrast, overproduction of mitochondrial ROS was rarely observed. Elevated ROS was found to be associated with lowered glutathione levels and depletion of antioxidant defense proteins. We also show for the first time that the levels of ROS generated were able to strongly promote the proliferation of AML cell lines, primary AML blasts, and, to a lesser extent, normal CD34(+) cells, and that the response to ROS is limited by the activation of the oxidative stress pathway mediated though p38(MAPK). Consistent with this, we observed that p38(MAPK) responses were attenuated in patients expressing high levels of ROS. These data show that overproduction of NOX-derived ROS can promote the proliferation of AML blasts and that they also develop mechanisms to suppress the stress signaling that would normally limit this response. Together these adaptations would be predicted to confer a competitive advantage to the leukemic clone.

  19. Human fetal mesenchymal stem cells.

    Science.gov (United States)

    O'Donoghue, Keelin; Chan, Jerry

    2006-09-01

    Stem cells have been isolated at all stages of development from the early developing embryo to the post-reproductive adult organism. However, the fetal environment is unique as it is the only time in ontogeny that there is migration of stem cells in large numbers into different organ compartments. While fetal neural and haemopoietic stem cells (HSC) have been well characterised, only recently have mesenchymal stem cells from the human fetus been isolated and evaluated. Our group have characterised in human fetal blood, liver and bone marrow a population of non-haemopoietic, non-endothelial cells with an immunophenotype similar to adult bone marrow-derived mesenchymal stem cells (MSC). These cells, human fetal mesenchymal stem cells (hfMSC), are true multipotent stem cells with greater self-renewal and differentiation capacity than their adult counterparts. They circulate in first trimester fetal blood and have been found to traffic into the maternal circulation, engrafting in bone marrow, where they remain microchimeric for decades after pregnancy. Though fetal microchimerism has been implicated in the pathogenesis of autoimmune disease, the biological role of hfMSC microchimerism is unknown. Potential downstream applications of hfMSC include their use as a target cell for non-invasive pre-natal diagnosis from maternal blood, and for fetal cellular and gene therapy. Using hfMSC in fetal therapy offers the theoretical advantages of avoidance of immune rejection, increased engraftment, and treatment before disease pathology sets in. Aside from allogeneic hfMSC in utero transplantation, the use of autologous hfMSC has been brought a step forward with the development of early blood sampling techniques, efficient viral transduction and clonal expansion. Work is ongoing to determine hfMSC fate post-transplantation in murine models of genetic disease. In this review we will examine what is known about hfMSC biology, as well as discussing areas for future research. The

  20. AML-loaded DC generate Th1-type cellular immune responses in vitro.

    Science.gov (United States)

    Xing, D; Decker, W K; Li, S; Robinson, S N; Yang, H; Segal, H; O'Connor, S; Yao, X; Komanduri, K V; McMannis, J D; Jones, R B; de Lima, M; Champlin, R E; Shpall, E J

    2006-01-01

    The generation of AML-specific T-lymphocyte responses by leukemia-derived DC has been documented by multiple investigators and is being pursued clinically. An obstacle to widespread use of this strategy is that it has not been possible to generate leukemic DC from all patients, and an alternative approach is needed if the majority of leukemia patients are to receive therapeutic vaccination in conjunction with other treatment protocols. In the present study, we generated DC from CD14-selected monocytes isolated from healthy donor PBPC and loaded them with a total cell lysate from AML patient blasts. Immature in vitro-derived DC exhibited robust phagocytic activity, and mature DC demonstrated high expression of CD80, CD83, CD86 and the chemokine receptor CCR7, important for DC migration to local lymph nodes. Mature, Ag-loaded DC were used as APC for leukemia-specific cytotoxic T-lymphocyte (CTL) induction and demonstrated cytotoxic activity against leukemic targets. CTL lysis was Ag-specific, with killing of both allogeneic leukemic blasts and autologous DC loaded with allogeneic AML lysate. HLA-matched controls were not lysed in our system. These data support further research into the use of this strategy as an alternative approach to leukemia-derived DC vaccination.

  1. Proportion of CD34 + CD38- Cell Population in Bone Marrow of Patients with De Novo AML as Prognostic Factor of Complete Rimission at First Course of Induction Chemotherapy%初诊急性髓系白血病骨髓CD34+CD38-细胞群比例是初次诱导缓解率的预后因素

    Institute of Scientific and Technical Information of China (English)

    张翠萍; 魏辉; 王慧君; 怀磊; 何侃; 陈一瑞; 林冬; 王建祥

    2011-01-01

    为了研究非M3急性髓系白血病(AML non-M3) CD34+ CD38 -细胞群及其G0期比例与临床和实验室特征的关系,使用流式细胞仪检测40例初治AML non-M3骨髓单个核细胞CD34和CD38的表达,测定各群细胞的细胞周期,并分析CD34+ CD38 -细胞群及其G0期比例与临床实验室特征及初次诱导缓解率的关系.结果显示,CD34+ CD38 -细胞群及其G0期的比例与染色体核型、初诊WBC计数及FLT3/ITD阳性均无明显相关性,但与诱导治疗后骨髓中幼稚细胞比例相关.诱导停疗第7天骨髓中有幼稚细胞患者CD34+ CD38-细胞比例为(12.47±26.26)%,诱导停疗第7天无幼稚细胞患者CD34+ CD38 -细胞比例为(2.62±7.20)% (p =0.031).诱导停疗第1天骨髓中可见幼稚细胞患者CD34+细胞群比例为(17.40±21.20)%,而诱导停疗第1天无幼稚细胞患者该比例为(5.64±6.96)%(p=0.00l).CR组患者治疗前CD34+ CD38 -细胞群的比例为(2.51±9.72)%,明显低于非CR组患者(24.92 +27.04%) (p =0.001).而在AMI non-M2b患者中,CR组患者治疗前CD34+ CD38 -细胞群的比例为(1.60±4.82)%,较非CR组患者更为降低(p <0.001).单因素分析显示,诱导化疗后是否取得CR与年龄(p=0.022)、CD34+ CD38 -细胞群比例(p=0.008)、诱导停疗第7天骨髓幼稚细胞比例(p=0.011)相关.多因素分析显示,仅有CD34+ CD38 -细胞群的比例与是否获得CR有相关趋势(p =0.052).结论:初治AML患者CD34+CD38 -细胞群的比例是AML初次诱导缓解率的预后因素.%This study was to investigate the relationship between the CD34* CD38 cell population and its proportion in Go phase of de novo AML non-M3 at diagnosis and the clinical and experimental characteristics. The flow cytometry was used to detect the expression of the cell surface antigen CD34 and CD38 in the bone marrow mononuclear cells (MNC) of the AML non-M, at diagnosis and investigate the cell cycle of the subpopulations, and then the relationships between the

  2. Differentiated human stem cells resemble fetal, not adult, β cells.

    Science.gov (United States)

    Hrvatin, Sinisa; O'Donnell, Charles W; Deng, Francis; Millman, Jeffrey R; Pagliuca, Felicia Walton; DiIorio, Philip; Rezania, Alireza; Gifford, David K; Melton, Douglas A

    2014-02-25

    Human pluripotent stem cells (hPSCs) have the potential to generate any human cell type, and one widely recognized goal is to make pancreatic β cells. To this end, comparisons between differentiated cell types produced in vitro and their in vivo counterparts are essential to validate hPSC-derived cells. Genome-wide transcriptional analysis of sorted insulin-expressing (INS(+)) cells derived from three independent hPSC lines, human fetal pancreata, and adult human islets points to two major conclusions: (i) Different hPSC lines produce highly similar INS(+) cells and (ii) hPSC-derived INS(+) (hPSC-INS(+)) cells more closely resemble human fetal β cells than adult β cells. This study provides a direct comparison of transcriptional programs between pure hPSC-INS(+) cells and true β cells and provides a catalog of genes whose manipulation may convert hPSC-INS(+) cells into functional β cells.

  3. AML1/RUNX1 Phosphorylation by Cyclin-Dependent Kinases Regulates the Degradation of AML1/RUNX1 by the Anaphase-Promoting Complex‡

    Science.gov (United States)

    Biggs, Joseph R.; Peterson, Luke F.; Zhang, Youhong; Kraft, Andrew S.; Zhang, Dong-Er

    2006-01-01

    AML1 (RUNX1) regulates hematopoiesis, angiogenesis, muscle function, and neurogenesis. Previous studies have shown that phosphorylation of AML1, particularly at serines 276 and 303, affects its transcriptional activation. Here, we report that phosphorylation of AML1 serines 276 and 303 can be blocked in vivo by inhibitors of the cyclin-dependent kinases (CDKs) Cdk1 and Cdk2. Furthermore, these residues can be phosphorylated in vitro by purified Cdk1/cyclin B and Cdk2/cyclin A. Mutant AML1 protein which cannot be phosphorylated at these sites (AML1-4A) is more stable than wild-type AML1. AML-4A is resistant to degradation mediated by Cdc20, one of the substrate-targeting subunits of the anaphase-promoting complex (APC). However, Cdh1, another targeting subunit used by the APC, can mediate the degradation of AML1-4A. A phospho-mimic protein, AML1-4D, can be targeted by Cdc20 or Cdh1. These observations suggest that both Cdc20 and Cdh1 can target AML1 for degradation by the APC but that AML1 phosphorylation may affect degradation mediated by Cdc20-APC to a greater degree. PMID:17015473

  4. Pro-apoptotic activity of α-bisabolol in preclinical models of primary human acute leukemia cells

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    Fato Romana

    2011-04-01

    Full Text Available Abstract Background We previously demonstrated that the plant-derived agent α-bisabolol enters cells via lipid rafts, binds to the pro-apoptotic Bcl-2 family protein BID, and may induce apoptosis. Here we studied the activity of α-bisabolol in acute leukemia cells. Methods We tested ex vivo blasts from 42 acute leukemias (14 Philadelphia-negative and 14 Philadelphia-positive B acute lymphoid leukemias, Ph-/Ph+B-ALL; 14 acute myeloid leukemias, AML for their sensitivity to α-bisabolol in 24-hour dose-response assays. Concentrations and time were chosen based on CD34+, CD33+my and normal peripheral blood cell sensitivity to increasing α-bisabolol concentrations for up to 120 hours. Results A clustering analysis of the sensitivity over 24 hours identified three clusters. Cluster 1 (14 ± 5 μM α-bisabolol IC50 included mainly Ph-B-ALL cells. AML cells were split into cluster 2 and 3 (45 ± 7 and 65 ± 5 μM IC50. Ph+B-ALL cells were scattered, but mainly grouped into cluster 2. All leukemias, including 3 imatinib-resistant cases, were eventually responsive, but a subset of B-ALL cells was fairly sensitive to low α-bisabolol concentrations. α-bisabolol acted as a pro-apoptotic agent via a direct damage to mitochondrial integrity, which was responsible for the decrease in NADH-supported state 3 respiration and the disruption of the mitochondrial membrane potential. Conclusion Our study provides the first evidence that α-bisabolol is a pro-apoptotic agent for primary human acute leukemia cells.

  5. Th17 cells and interleukin-17 increase with poor prognosis in patients with acute myeloid leukemia.

    Science.gov (United States)

    Han, Yixiang; Ye, Aifang; Bi, Laixi; Wu, Jianbo; Yu, Kang; Zhang, Shenghui

    2014-08-01

    Although Th17 cells play crucial roles in the pathogenesis of many autoimmune and inflammatory disorders, their roles in malignancies are currently under debate. The role and mechanism of Th17 cells in patients with acute myeloid leukemia (AML) remain poorly understood. Here we demonstrated that the frequency of Th17 cells was significantly increased in peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells from AML patients compared with healthy donors. Plasma levels of interleukin (IL)-17, IL-22, IL-23, IL-1β, IL-6, and transforming growth factor (TGF)-β1 were significantly increased in blood and bone marrow in AML patients compared with healthy donors. The in vitro experiments demonstrated that IL-1β, IL-6, IL-23, but not TGF-β1 promoted the generation and differentiation of Th17 cells from naive CD4(+) T cells in humans. IL-17A, a signature cytokine secreted by Th17 cells, induced the proliferation of IL-17 receptor (IL-17R)-positive AML cells via IL-17R, in which activation of PI3K/Akt and Jak/Stat3 signaling pathway may play important roles. In addition, combination of IL-17A and IL-22 significantly reduced the generation of Th1 cells and the production of interferon (IFN)-γ from healthy donor or AML patient peripheral blood mononuclear cells. Patients with high Th17 cell frequency had poor prognosis, whereas patients with high Th1 cell frequency had prolonged survival. Combined analysis of Th1 and Th17 cell frequencies improved the ability to predict patient outcomes. In conclusion, Th17 cells play a crucial role in the pathogenesis of AML and may be an important therapeutic target and prognostic predictor.

  6. The potency of human testicular stem cells

    NARCIS (Netherlands)

    Chikhovskaya, J.V.

    2013-01-01

    In this thesis, we evaluate the stem cell state of cells present in primary human testicular cell cultures as well as their origin and relation to germ or somatic lineages within testicular tissue. We conclude that human testis-derived embryonic stem cell-like (htES-like) colonies arising in primary

  7. Apoptosis-inducing activity of Helleborus niger in ALL and AML.

    Science.gov (United States)

    Jesse, Patrick; Mottke, Gritt; Eberle, Jürgen; Seifert, Georg; Henze, Günter; Prokop, Aram

    2009-04-01

    Helleborus niger is used in the adjuvant treatment of different tumors in anthroposophical medicine. Indications include various types of brain tumors in children, as well as prostate cancer, leukemia and lymphoma. Our aim was to investigate the therapeutic effects of these extracts apart from the traditional use. : We used an aqueous whole plant extract of H. niger in different cancer and leukemia cell lines and primary cells of patients with childhood ALL and AML and identified the main mechanisms of action. A strong inhibition of proliferation is caused by specific apoptosis induction, which is executed via the mitochondrial pathway and caspase-3 processing. Apoptosis could be detected in lymphoma (BJAB), leukemia (Reh, Nalm6, Sup-B15) and melanoma (Mel-HO) cells and overcomes a Bcl-2-mediated block of apoptosis. In primary cells of patients with childhood ALL and AML, which were partly poor responding to doxorubicin and daunorubicin, a strong apoptosis induction was determined. In combination with the vinca alkaloid vincristine, strong synergistic effects were detected in BJAB cells. We demonstrate in vitro efficacy of H. niger extract in cells of hematological malignancies; these studies should encourage in vivo experiments. Copyright 2008 Wiley-Liss, Inc.

  8. Stem cell differentiation and human liver disease

    Institute of Scientific and Technical Information of China (English)

    Wen-Li Zhou; Claire N Medine; Liang Zhu; David C Hay

    2012-01-01

    Human stem cells are scalable cell populations capable of cellular differentiation.This makes them a very attractive in vitro cellular resource and in theory provides unlimited amounts of primary cells.Such an approach has the potential to improve our understanding of human biology and treating disease.In the future it may be possible to deploy novel stem cell-based approaches to treat human liver diseases.In recent years,efficient hepatic differentiation from human stem cells has been achieved by several research groups including our own.In this review we provide an overview of the field and discuss the future potential and limitations of stem cell technology.

  9. Leukemia-associated NF1 inactivation in patients with pediatric T-ALL and AML lacking evidence for neurofibromatosis.

    Science.gov (United States)

    Balgobind, Brian V; Van Vlierberghe, Pieter; van den Ouweland, Ans M W; Beverloo, H Berna; Terlouw-Kromosoeto, Joan N R; van Wering, Elisabeth R; Reinhardt, Dirk; Horstmann, Martin; Kaspers, Gertjan J L; Pieters, Rob; Zwaan, C Michel; Van den Heuvel-Eibrink, Marry M; Meijerink, Jules P P

    2008-04-15

    Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder caused by mutations in the NF1 gene. Patients with NF1 have a higher risk to develop juvenile myelomonocytic leukemia (JMML) with a possible progression toward acute myeloid leukemia (AML). In an oligo array comparative genomic hybridization-based screening of 103 patients with pediatric T-cell acute lymphoblastic leukemia (T-ALL) and 71 patients with MLL-rearranged AML, a recurrent cryptic deletion, del(17)(q11.2), was identified in 3 patients with T-ALL and 2 patients with MLL-rearranged AML. This deletion has previously been described as a microdeletion of the NF1 region in patients with NF1. However, our patients lacked clinical NF1 symptoms. Mutation analysis in 4 of these del(17)(q11.2)-positive patients revealed that mutations in the remaining NF1 allele were present in 3 patients, confirming its role as a tumor-suppressor gene in cancer. In addition, NF1 inactivation was confirmed at the RNA expression level in 3 patients tested. Since the NF1 protein is a negative regulator of the RAS pathway (RAS-GTPase activating protein), homozygous NF1 inactivation represent a novel type I mutation in pediatric MLL-rearranged AML and T-ALL with a predicted frequency that is less than 10%. NF1 inactivation may provide an additional proliferative signal toward the development of leukemia.

  10. The human airway epithelial basal cell transcriptome.

    Directory of Open Access Journals (Sweden)

    Neil R Hackett

    Full Text Available BACKGROUND: The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population. METHODOLOGY/PRINCIPAL FINDINGS: Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the "human airway basal cell signature" as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels. CONCLUSION/SIGNIFICANCE: The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of

  11. c-Myc regulates expression of NKG2D ligands ULBP1/2/3 in AML and modulates their susceptibility to NK-mediated lysis.

    Science.gov (United States)

    Nanbakhsh, Arash; Pochon, Cécile; Mallavialle, Aude; Amsellem, Sophie; Bourhis, Jean Henri; Chouaib, Salem

    2014-06-05

    Cytarabine (cytosine arabinoside) is one of the most effective drugs for the treatment of patients diagnosed with acute myeloid leukemia (AML). Despite its efficiency against AML cells, the emergence of drug resistance due to prolonged chemotherapy in most patients is still a major obstacle. Several studies have shown that drug resistance mechanisms alter the sensitivity of leukemia cells to immune system effector cells. To investigate this phenomenon, parental acute myeloid cell lines, HL-60 and KG-1, were continuously exposed to increasing doses of cytarabine in order to establish equivalent resistant cell lines, HL-60(R) and KG-1(R). Our data indicate that cytarabine-resistant cells are more susceptible to natural killer (NK)-mediated cell lysis as compared with parental cytarabine-sensitive cells. The increased susceptibility correlates with the induction of UL-16 binding proteins (ULBP) 1/2/3 and NK group 2, member D (NKG2D) ligands on target cells by a mechanism involving c-Myc induction. More importantly, chromatin immunoprecipitation assay revealed that ULBP1/3 are direct targets of c-Myc. Using drug-resistant primary AML blasts as target cells, inhibition of c-Myc resulted in decreased expression of NKG2D ligands and the subsequent impairment of NK cell lysis. This study provides for the first time, the c-Myc dependent regulation of NKG2D ligands in AML. © 2014 by The American Society of Hematology.

  12. Therapeutic Effects of Myeloid Cell Leukemia-1 siRNA on Human Acute Myeloid Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Hadi Karami

    2014-05-01

    Full Text Available Purpose: Up-regulation of Mcl-1, a known anti-apoptotic protein, is associated with the survival and progression of various malignancies including leukemia. The aim of this study was to explore the effect of Mcl-1 small interference RNA (siRNA on the proliferation and apoptosis of HL-60 acute myeloid leukemia (AML cells. Methods: siRNA transfection was performed using Lipofectamine™2000 reagent. Relative mRNA and protein expressions were quantified by quantitative real-time PCR and Western blotting, respectively. Trypan blue assay was performed to assess tumor cell proliferation after siRNA transfection. The cytotoxic effect of Mcl-1 siRNA on leukemic cells was measured using MTT assay. Apoptosis was detected using ELISA cell death assay. Results: Mcl-1 siRNA clearly lowered both Mcl-1 mRNA and protein levels in a time-dependent manner, leading to marked inhibition of cell survival and proliferation. Furthermore, Mcl-1 down-regulation significantly enhanced the extent of HL-60 apoptotic cells. Conclusion: Our results suggest that the down-regulation of Mcl-1 by siRNA can effectively trigger apoptosis and inhibit the proliferation of leukemic cells. Therefore, Mcl-1 siRNA may be a potent adjuvant in AML therapy.

  13. The Runt domain of AML1 (RUNX1) binds a sequence-conserved RNA motif that mimics a DNA element

    Science.gov (United States)

    Fukunaga, Junichi; Nomura, Yusuke; Tanaka, Yoichiro; Amano, Ryo; Tanaka, Taku; Nakamura, Yoshikazu; Kawai, Gota; Sakamoto, Taiichi; Kozu, Tomoko

    2013-01-01

    AML1 (RUNX1) is a key transcription factor for hematopoiesis that binds to the Runt-binding double-stranded DNA element (RDE) of target genes through its N-terminal Runt domain. Aberrations in the AML1 gene are frequently found in human leukemia. To better understand AML1 and its potential utility for diagnosis and therapy, we obtained RNA aptamers that bind specifically to the AML1 Runt domain. Enzymatic probing and NMR analyses revealed that Apt1-S, which is a truncated variant of one of the aptamers, has a CACG tetraloop and two stem regions separated by an internal loop. All the isolated aptamers were found to contain the conserved sequence motif 5′-NNCCAC-3′ and 5′-GCGMGN′N′-3′ (M:A or C; N and N′ form Watson–Crick base pairs). The motif contains one AC mismatch and one base bulged out. Mutational analysis of Apt1-S showed that three guanines of the motif are important for Runt binding as are the three guanines of RDE, which are directly recognized by three arginine residues of the Runt domain. Mutational analyses of the Runt domain revealed that the amino acid residues used for Apt1-S binding were similar to those used for RDE binding. Furthermore, the aptamer competed with RDE for binding to the Runt domain in vitro. These results demonstrated that the Runt domain of the AML1 protein binds to the motif of the aptamer that mimics DNA. Our findings should provide new insights into RNA function and utility in both basic and applied sciences. PMID:23709277

  14. Endocannabinoids and Human Sperm Cells

    Directory of Open Access Journals (Sweden)

    Giovanna Zolese

    2010-10-01

    Full Text Available N-acylethanolamides (NAEs are naturally occurring signaling lipids consisting of amides and esters of long-chain polyunsaturated fatty acids. Usually they are present in a very small amounts in many mammalian tissues and cells, including human reproductive tracts and fluids. Recently, the presence of N-arachidonoylethanolamide (anandamide, AEA, the most characterised member of endocannabinoids, and its congeners palmitoylethanolamide (PEA and oleylethanolamide (OEA in seminal plasma, oviductal fluid, and follicular fluids was demonstrated. AEA has been shown to bind not only type-1 (CB1 and type-2 (CB2 cannabinoid receptors, but also type-1 vanilloid receptor (TRPV1, while PEA and OEA are inactive with respect to classical cannabinoid CB1 and CB2 but activate TRPV1 or peroxisome proliferator activate receptors (PPARs. This review concerns the most recent experimental data on PEA and OEA, endocannabinoid-like molecules which appear to exert their action exclusively on sperm cells with altered features, such as membrane characteristics and kinematic parameters. Their beneficial effects on these cells could suggest a possible pharmacological use of PEA and OEA on patients affected by some forms of idiopathic infertility.

  15. Human regulatory B cells control the TFH cell response.

    Science.gov (United States)

    Achour, Achouak; Simon, Quentin; Mohr, Audrey; Séité, Jean-François; Youinou, Pierre; Bendaoud, Boutahar; Ghedira, Ibtissem; Pers, Jacques-Olivier; Jamin, Christophe

    2017-07-01

    Follicular helper T (TFH) cells support terminal B-cell differentiation. Human regulatory B (Breg) cells modulate cellular responses, but their control of TFH cell-dependent humoral immune responses is unknown. We sought to assess the role of Breg cells on TFH cell development and function. Human T cells were polyclonally stimulated in the presence of IL-12 and IL-21 to generate TFH cells. They were cocultured with B cells to induce their terminal differentiation. Breg cells were included in these cultures, and their effects were evaluated by using flow cytometry and ELISA. B-cell lymphoma 6, IL-21, inducible costimulator, CXCR5, and programmed cell death protein 1 (PD-1) expressions increased on stimulated human T cells, characterizing TFH cell maturation. In cocultures they differentiated B cells into CD138(+) plasma and IgD(-)CD27(+) memory cells and triggered immunoglobulin secretions. Breg cells obtained by Toll-like receptor 9 and CD40 activation of B cells prevented TFH cell development. Added to TFH cell and B-cell cocultures, they inhibited B-cell differentiation, impeded immunoglobulin secretions, and expanded Foxp3(+)CXCR5(+)PD-1(+) follicular regulatory T cells. Breg cells modulated IL-21 receptor expressions on TFH cells and B cells, and their suppressive activities involved CD40, CD80, CD86, and intercellular adhesion molecule interactions and required production of IL-10 and TGF-β. Human Breg cells control TFH cell maturation, expand follicular regulatory T cells, and inhibit the TFH cell-mediated antibody secretion. These novel observations demonstrate a role for the Breg cell in germinal center reactions and suggest that deficient activities might impair the TFH cell-dependent control of humoral immunity and might lead to the development of aberrant autoimmune responses. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  16. A new model to predict remission status in AML patients based on day 14 bone marrow biopsy.

    Science.gov (United States)

    Norkin, Maxim; Chang, Myron; An, Qi; Leather, Helen; Katragadda, Lakshmikanth; Li, Ying; Moreb, Jan S; May, W Stratford; Brown, Randy A; Hsu, Jack W; Hiemenz, John W; Wingard, John R; Cogle, Christopher R

    2016-07-01

    Although bone marrow evaluation on day 14 after initiation of induction chemotherapy (D14 BM) is a widely accepted practice in patients with acute myeloid leukemia (AML), it has suboptimal predictive value for predicting complete remission. We retrospectively analyzed pretreatment characteristics and post-induction response in a cohort of AML patients to determine if adding clinical and laboratory characteristics can improve the predictive value of the D14 BM evaluation. Among 297 patients treated for AML at the single institution 183 patients (61%) had leukemia-positive D14 BM. Of those, 94 were given reinduction chemotherapy and 89 were not. Of the 89 patients who did not receive reinduction, 32 (36%) subsequently achieved complete remission (CR) or complete remission with incomplete count recovery (CRi), and 57 (64%) had persistent disease. Persistent disease after positive D14 BM was more likely associated with higher percentage of D14 myeloblasts, a history of relapsed disease before induction, and higher risk disease compared to patients who subsequently achieved CR. Age, diagnostic white blood cell count, and the D14 BM cellularity did not influence the subsequent likelihood of achieving remission in patients with a positive D14 BM. A new mathematical equation was created and resulted in a positive predictive value of 83%, negative predictive value 90% and accuracy 88% for correctly identifying remission status after positive D14 BM in AML. The accuracy of predicting response using these additional parameters was significantly higher than without (0.88 vs. 0.80, P=0.002). Our new model provides better accuracy for predicting the likelihood of achieving remission and if validated in future studies may be useful for managing AML patients. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Core-binding factor acute myeloid leukemia in first relapse: a retrospective study from the French AML Intergroup.

    Science.gov (United States)

    Hospital, Marie-Anne; Prebet, Thomas; Bertoli, Sarah; Thomas, Xavier; Tavernier, Emmanuelle; Braun, Thorsten; Pautas, Cécile; Perrot, Aurore; Lioure, Bruno; Rousselot, Philippe; Tamburini, Jérôme; Cluzeau, Thomas; Konopacki, Johanna; Randriamalala, Edouard; Berthon, Céline; Gourin, Marie-Pierre; Recher, Christian; Cahn, Jean-Yves; Ifrah, Norbert; Dombret, Hervé; Boissel, Nicolas

    2014-08-21

    Although core-binding factor-acute myeloid leukemia (CBF-AML) (t[8;21] or inv[16]/t[16;16]) represents a favorable cytogenetic AML subgroup, 30% to 40% of these patients relapse after standard intensive chemotherapy. The encouraging results of gemtuzumab ozogamicin (GO) in newly diagnosed AML, and particularly in CBF-AML, incited us to retrospectively investigate the impact of GO-based salvage in these patients. We retrospectively analyzed the outcome of 145 patients with CBF-AML (59 t[8;21], 86 inv[16]/t[16;16]) in first relapse. As salvage, 48 patients received GO-based chemotherapy and 97 patients received conventional chemotherapy. Median age was 43 years (range, 16-76). Median first complete remission duration was 12.1 months (range, 2.1-93.6). Overall, second complete remission (CR2) rate was 88%. With a median follow-up from relapse of 3.5 years, the estimated 5-year disease-free survival (DFS) was 50% and 5-year overall survival (OS) was 51%. Older age and shorter first complete remission duration was associated with a shorter OS. Patients treated with GO had similar CR2 rate but significantly higher 5-year DFS (68% vs 42%; P = .05) and OS (65% vs 44%; P = .02). In multivariate analysis, GO salvage was still associated with a significant benefit in DFS and OS. In the 78 patients who received allogeneic hematopoietic stem cell transplantation in CR2, GO before transplant significantly improved posttransplant DFS and OS without excess of treatment-related mortality.

  18. Identification of TEL-AML1 (ETV6-RUNX1 associated DNA and its impact on mRNA and protein output using ChIP, mRNA expression arrays and SILAC

    Directory of Open Access Journals (Sweden)

    Yvonne Linka

    2014-12-01

    Full Text Available The contribution of the most common reciprocal translocation in childhood B-cell precursor leukemia t(12;21(p13;q22 to leukemia development is still under debate. Direct as well as secondary indirect effects of the TEL-AML1 fusion protein are commonly recorded by using cell lines and patient samples, often bearing the TEL-AML1 fusion protein for decades. To identify direct targets of the fusion protein a short-term induction of TEL-AML1 is needed. We here describe in detail the experimental procedure, quality controls and contents of the ChIP, mRNA expression and SILAC datasets associated with the study published by Linka and colleagues in the Blood Cancer Journal [1] utilizing a short term induction of TEL-AML1 in an inducible precursor B-cell line model.

  19. Satellite cells in human skeletal muscle plasticity.

    Science.gov (United States)

    Snijders, Tim; Nederveen, Joshua P; McKay, Bryon R; Joanisse, Sophie; Verdijk, Lex B; van Loon, Luc J C; Parise, Gianni

    2015-01-01

    Skeletal muscle satellite cells are considered to play a crucial role in muscle fiber maintenance, repair and remodeling. Our knowledge of the role of satellite cells in muscle fiber adaptation has traditionally relied on in vitro cell and in vivo animal models. Over the past decade, a genuine effort has been made to translate these results to humans under physiological conditions. Findings from in vivo human studies suggest that satellite cells play a key role in skeletal muscle fiber repair/remodeling in response to exercise. Mounting evidence indicates that aging has a profound impact on the regulation of satellite cells in human skeletal muscle. Yet, the precise role of satellite cells in the development of muscle fiber atrophy with age remains unresolved. This review seeks to integrate recent results from in vivo human studies on satellite cell function in muscle fiber repair/remodeling in the wider context of satellite cell biology whose literature is largely based on animal and cell models.

  20. Monitoring response and resistance to the novel arsenical darinaparsin in an AML patient

    Directory of Open Access Journals (Sweden)

    Torsten Holm eNielsen

    2013-02-01

    Full Text Available Acute myeloid leukemia (AML with inversion of chromosome 3 is characterized by overexpression of EVI1 and carries a dismal prognosis. Arsenic-containing compounds have been described to be efficacious in malignancies overexpressing EVI1. Here we describe a case of AML with inv(3(q21q26.2 treated with the organic arsenical darinaparsin. Using a personalized medicine approach, different arsenicals were screened for anti-leukemic effect against the patient’s cells ex vivo. The most promising compound, darinaparsin, was selected for in vivo treatment. Clinical effect was almost immediate, with a normalization of temperature, a stabilization of white blood cell (WBC counts and an increased quality of life. Longitudinal monitoring of patient response and resistance incorporating significant correlative studies on patient derived blood samples over the two cycles of darinaparsin given to this patient allowed us to evaluate potential mechanisms of response and resistance. The anti-leukemic effects of darinaparsin correlated with inhibition of the alternative NF-κB pathway and production of the inflammatory cytokine IL-8. Emergence of resistance was suspected during treatment cycle 2 and supported by xenograft studies in nude mice. Darinaparsin resistance correlated with an attenuation of the effect of treatment on the alternative NF-κB pathway. The results from this patient indicate that darinaparsin may be a good treatment option for inv(3 AML and that inhibition of the alternative NF-κB pathway may be predictive of response. Longitudinal monitoring of disease response as well as several correlative parameters allowed for the generation of novel correlations and predictors of response to experimental therapy in a heavily pretreated patient.

  1. Induced Mitogenic Activity in AML-12 Mouse Hepatocytes Exposed to Low-dose Ultra-Wideband Electromagnetic Radiation

    Directory of Open Access Journals (Sweden)

    P. B. Tchounwou

    2005-04-01

    Full Text Available Ultra–wideband (UWB technology has increased with the use of various civilian and military applications. In the present study, we hypothesized that low-dose UWB electromagnetic radiation (UWBR could elicit a mitogenic effect in AML-12 mouse hepatocytes, in vitro. To test this hypothesis, we exposed AML-12 mouse hepatocytes, to UWBR in a specially constructed gigahertz transverse electromagnetic mode (GTEM cell. Cells were exposed to UWBR for 2 h at a temperature of 23°C, a pulse width of 10 ns, a repetition rate of 1 kHz, and field strength of 5-20 kV/m. UWB pulses were triggered by an external pulse generator for UWBR exposure but were not triggered for the sham exposure. We performed an MTT Assay to assess cell viability for UWBR-treated and sham-exposed hepatocytes. Data from viability studies indicated a time-related increase in hepatocytes at time intervals from 8-24 h post exposure. UWBR exerted a statistically significant (p < 0.05 dose-dependent response in cell viability in both serum-treated and serum free medium (SFM -treated hepatocytes. Western blot analysis of hepatocyte lysates demonstrated that cyclin A protein was induced in hepatocytes, suggesting that increased MTT activity after UWBR exposure was due to cell proliferation. This study indicates that UWBR has a mitogenic effect on AML-12 mouse hepatocytes and implicates a possible role for UWBR in hepatocarcinoma.

  2. Acute myeloid leukemia targeting by myxoma virus in vivo depends on cell binding but not permissiveness to infection in vitro.

    Science.gov (United States)

    Madlambayan, Gerard J; Bartee, Eric; Kim, Manbok; Rahman, Masmudur M; Meacham, Amy; Scott, Edward W; McFadden, Grant; Cogle, Christopher R

    2012-05-01

    Some oncolytic viruses, such as myxoma virus (MYXV), can selectively target malignant hematopoietic cells, while sparing normal hematopoietic cells. This capacity for discrimination creates an opportunity to use oncolytic viruses as ex vivo purging agents of autologous hematopoietic cell grafts in patients with hematologic malignancies. However, the mechanisms by which oncolytic viruses select malignant hematopoietic cells are poorly understood. In this study, we investigated how MYXV specifically targets human AML cells. MYXV prevented chloroma formation and bone marrow engraftment of two human AML cell lines, KG-1 and THP-1. The reduction in human leukemia engraftment after ex vivo MYXV treatment was dose-dependent and required a minimum MOI of 3. Both AML cell lines demonstrated MYXV binding to leukemia cell membranes following co-incubation: however, evidence of productive MYXV infection was observed only in THP-1 cells. This observation, that KG-1 can be targeted in vivo even in the absence of in vitro permissive viral infection, contrasts with the current understanding of oncolytic virotherapy, which assumes that virus infection and productive replication is a requirement. Preventing MYXV binding to AML cells with heparin abrogated the purging capacity of MYXV, indicating that binding of infectious virus particles is a necessary step for effective viral oncolysis. Our results challenge the current dogma of oncolytic virotherapy and show that in vitro permissiveness to an oncolytic virus is not necessarily an accurate predictor of oncolytic potency in vivo.

  3. Activated leukemic oncogenes AML1-ETO and c-kit: role in development of acute myeloid leukemia and current approaches for their inhibition.

    Science.gov (United States)

    Rulina, A V; Spirin, P V; Prassolov, V S

    2010-12-01

    Acute myeloid leukemia (AML) is a malignant blood disease caused by different mutations that enhance the proliferative activity and survival of blood cells and affect their differentiation and apoptosis. The most frequent disorders in AML are translocations between chromosomes 21 and 8 leading to production of a chimeric oncogene, AML1-ETO, and hyperexpression of the receptor tyrosine kinase KIT. Mutations in these genes often occur jointly. The presence in cells of two activated oncogenes is likely to trigger their malignization. The current approaches for treatment of oncologic diseases (bone marrow transplantation, radiotherapy, and chemotherapy) have significant shortcomings, and thus many laboratories are intensively developing new approaches against leukemias. Inhibiting expression of activated leukemic oncogenes based on the principle of RNA interference seems to be a promising approach in this field.

  4. IMMUNORESPONSES OF HUMANIZED SCID MICE TO HUMAN LUNG CANCER CELLS

    Institute of Scientific and Technical Information of China (English)

    陈力真; 王树蕙; 张云; 王世真

    1996-01-01

    HuPBL-SCID mice were used to explore how they would response to human ttmoor cells of 801/MLC.Living 801/MLC cells appeared to be fetal to the the mice due to the production of human TNF. The huP-BL-SCID rniee did not generate any noticeable amotmt of specific human immunoglobttlin either by single immunization with living 801/MLC cells or by repeated immunization with irradiated 801/MLC cells. Our preliminary experiments with huPBL-SCID mice showed that such chimeras would he a very useful models for tumor immunological researches.

  5. Search for naive human pluripotent stem cells

    Institute of Scientific and Technical Information of China (English)

    Simone Aparecida Siqueira Fonseca; Roberta Montero Costas; Lygia Veiga Pereira

    2015-01-01

    Normal mouse pluripotent stem cells were originallyderived from the inner cell mass (ICM) of blastocystsand shown to be the in vitro equivalent of those preimplantationembryonic cells, and thus were calledembryonic stem cells (ESCs). More than a decade later,pluripotent cells were isolated from the ICM of humanblastocysts. Despite being called human ESCs, thesecells differ significantly from mouse ESCs, includingdifferent morphology and mechanisms of control ofpluripotency, suggesting distinct embryonic originsof ESCs from the two species. Subsequently, mousepluripotent stem cells were established from the ICMderivedepiblast of post-implantation embryos. Thesemouse epiblast stem cells (EpiSCs) are morphologicaland epigenetically more similar to human ESCs. Thisraised the question of whether cells from the humanICM are in a more advanced differentiation stage thantheir murine counterpart, or whether the availableculture conditions were not adequate to maintain thosehuman cells in their in vivo state, leading to a transitioninto EpiSC-like cells in vitro . More recently, novel cultureconditions allowed the conversion of human ESCs intomouse ESC-like cells called naive (or ground state)human ESCs, and the derivation of naive human ESCsfrom blastocysts. Here we will review the characteristicsof each type of pluripotent stem cells, how (andwhether) these relate to different stages of embryonicdevelopment, and discuss the potential implications ofnaive human ESCs in research and therapy.

  6. Treatment results of three consecutive German childhood AML trials: BFM-78, -83, and -87. AML-BFM-Group.

    Science.gov (United States)

    Ritter, J; Creutzig, U; Schellong, G

    1992-01-01

    Acute myelogenous leukemia (AML) represents a heterogenous group of leukemias in adults as well as in children. The BFM group initiated 3 consecutive studies on the treatment of this disease. Between December 1978 and April 1991, 543 children under the age of 17 years entered the 3 consecutive multicenter studies, AML-BFM-78 (n = 151), AML-BFM-83 (n = 182), and the still ongoing study AML-BFM-87 (n = 210). The treatment strategy of BFM-78 consisted of an eight week induction/-consolidation regimen employing 7 different drugs together with cranial irradiation, followed by continuous maintenance for two years. The main alteration in the second study BFM-83 was the addition of an intensive 8-day ADE induction course (cytosine arabinoside, daunorubicin, etoposide). In the ongoing trial BFM-87 two courses of HD-ARA-C and etoposide are given after consolidation. CR rates were 80% in trials I and II, and 78% in trial III. The probability of a 4.5-year event-free survival was 35%, SD 4% in study I; 49%, SD 4% in study II, and 45%, SD 4% in study III. The probability of a 4.5-year event-free interval (EFI) was increased from 45%, SD 5% in study I to 61%, SD 4% in study II, it is in the same range in study III (58%, SD 5%). Seven of 10 children which underwent bone marrow transplantation (BMT) in 1. CR are still in first CR after a maximum follow-up time of 3.5 yrs. In summary, the addition of HD-ARA-C together with etoposide given after induction/consolidation treatment did not further reduce the incidence of relapses in childhood AML. So far, the results of study BFM-87 are in the same range than those of study BFM-83.

  7. Endothelial cells derived from human embryonic stem cells

    Science.gov (United States)

    Levenberg, Shulamit; Golub, Justin S.; Amit, Michal; Itskovitz-Eldor, Joseph; Langer, Robert

    2002-04-01

    Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

  8. Trisomy 8 in Pediatric Acute Myeloid Leukemia. A NOPHO-AML Study

    DEFF Research Database (Denmark)

    Laursen, Anne Cathrine Lund; Sandahl, Julie Damgaard; Kjeldsen, Eigil

    2016-01-01

    Trisomy 8 (+8) is a common cytogenetic aberration in acute myeloid leukemia (AML); however, the impact of +8 in pediatric AML is largely unknown. We retrospectively investigated 609 patients from the NOPHO-AML database to determine the clinical and cytogenetic characteristics of +8 in pediatric AML......;11). Trisomy 8 alone was associated with older age (median age 10.1 years), FAB M2 (33%), and FLT3-ITD mutations (58%). The 5-year event-free survival for patients with +8 alone was 50% and 5-year overall survival was 75%. In conclusion, +8 is one of the most common cytogenetic aberrations in pediatric AML...

  9. Induced pluripotent stem cell lines derived from human somatic cells.

    Science.gov (United States)

    Yu, Junying; Vodyanik, Maxim A; Smuga-Otto, Kim; Antosiewicz-Bourget, Jessica; Frane, Jennifer L; Tian, Shulan; Nie, Jeff; Jonsdottir, Gudrun A; Ruotti, Victor; Stewart, Ron; Slukvin, Igor I; Thomson, James A

    2007-12-21

    Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

  10. Trophoblast lineage cells derived from human induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ying, E-mail: ying.chen@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Wang, Kai; Chandramouli, Gadisetti V.R. [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Knott, Jason G. [Developmental Epigenetics Laboratory, Department of Animal Science, Michigan State University (United States); Leach, Richard, E-mail: Richard.leach@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Department of Obstetrics, Gynecology and Women’s Health, Spectrum Health Medical Group (United States)

    2013-07-12

    Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.

  11. The molecular heterogenity of MLL-rearranged pediatric AML

    NARCIS (Netherlands)

    E.A. Coenen (Eva)

    2013-01-01

    textabstractContinuous renewal of blood cells, so called hematopoiesis, is essential for human life. Starting from several months after birth the majority of blood cell production occurs in the bone marrow. Circulating blood cells are of diverse morphology and function and can be divided in red bloo

  12. Sensitivity of acute myeloid leukemia Kasumi-1 cells to binase toxic action depends on the expression of KIT and АML1-ETO oncogenes.

    Science.gov (United States)

    Mitkevich, Vladimir A; Petrushanko, Irina Y; Spirin, Pavel V; Fedorova, Tatiana V; Kretova, Olga V; Tchurikov, Nickolai A; Prassolov, Vladimir S; Ilinskaya, Olga N; Makarov, Alexander A

    2011-12-01

    Some RNases selectively attack malignant cells, triggering an apoptotic response, and therefore are considered as alternative chemotherapeutic drugs. Here we studied the effects of Bacillus intermedius RNase (binase) on murine myeloid progenitor cells FDC-P1; transduced FDC-P1 cells ectopically expressing mutated human KIT N822K oncogene and/or human AML1-ETO oncogene; and human leukemia Kasumi-1 cells expressing both of these oncogenes. Expression of both KIT and AML1-ETO oncogenes makes FDC-P1 cells sensitive to the toxic effects of binase. Kasumi-1 cells were the most responsive to the toxic actions of binase among the cell lines used in this work with an IC50 value of 0.56 µM. Either blocking the functional activity of the KIT protein with imatinib or knocking-down oncogene expression using lentiviral vectors producing shRNA against AML1-ETO or KIT eliminated the sensitivity of Kasumi-1 cells to binase toxic action and promoted their survival, even in the absence of KIT-dependent proliferation and antiapoptotic pathways. Here we provide evidence that the cooperative effect of the expression of mutated KIT and AML1-ETO oncogenes is crucial for selective toxic action of binase on malignant cells. These findings can facilitate clinical applications of binase providing a useful screen based on the presence of the corresponding target oncogenes in malignant cells.

  13. Human Neural Cell-Based Biosensor

    Science.gov (United States)

    2013-05-28

    including incubation with factors such as SHH ) and proceed to Human Neural Progenitor Cells Dopaminergic Differentiation β-III Tubulin/TH...exposure in human embryonic stem cells. J Recept Signal Transduct Res. 2011 Jun;31(3):206-13. Gerwe BA, Angel PM, West FD, Hasneen K, Young A

  14. Study of Rpl22 in MDS and AML

    Science.gov (United States)

    2015-10-01

    usually linked with bone marrow disease , our data doesn’t support p53 is the key player in MDS/AML caused by Rpl22 defect. 3. Uncovered a pathological...our lab and others supports that ribosomal proteins play a critical role in development(1) and diseases including bone marrow disease (2) in addition...that RP play a critical but poorly understood role in development as well as disease . Mutations in RP cause a group of diseases collectively termed

  15. [Research progress of epigenetic drug decitabine in AML].

    Science.gov (United States)

    Zhang, Rui; Li, Hui-Min

    2014-10-01

    Epigenetics is a gene regulation mechanism that can be reversible and heritable, but do not involve the DNA sequence changes. DNA methylation is one of the most important epigenetic modifications, which is closely correlate with tumorigenesis. Decitabine is a methylation inhibitor, which has different action mechanism and targeting characteristics from the traditional chemotherapy, representing a new therapeutic strategy. This review mainly focuses on the anti-leukemia mechanism of decitabine and its clinical efficacy for AML.

  16. Characterization of miRNomes in Acute and Chronic Myeloid Leukemia Cell Lines

    Institute of Scientific and Technical Information of China (English)

    Qian Xiong; Jiangwei Yan; Songnian Hu; Xiangdong Fang; Yadong Yang; Hai Wang; Jie Li; Shaobin Wang; Yanming Li; Yaran Yang; Kan Cai; Xiuyan Ruan

    2014-01-01

    Myeloid leukemias are highly diverse diseases and have been shown to be associated with microRNA (miRNA) expression aberrations. The present study involved an in-depth miRNome analysis of two human acute myeloid leukemia (AML) cell lines, HL-60 and THP-1, and one human chronic myeloid leukemia (CML) cell line, K562, via massively parallel signature sequenc-ing. mRNA expression profiles of these cell lines that were established previously in our lab facil-itated an integrative analysis of miRNA and mRNA expression patterns. miRNA expression profiling followed by differential expression analysis and target prediction suggested numerous miRNA signatures in AML and CML cell lines. Some miRNAs may act as either tumor suppres-sors or oncomiRs in AML and CML by targeting key genes in AML and CML pathways. Expres-sion patterns of cell type-specific miRNAs could partially reflect the characteristics of K562, HL-60 and THP-1 cell lines, such as actin filament-based processes, responsiveness to stimulus and phag-ocytic activity. miRNAs may also regulate myeloid differentiation, since they usually suppress dif-ferentiation regulators. Our study provides a resource to further investigate the employment of miRNAs in human leukemia subtyping, leukemogenesis and myeloid development. In addition, the distinctive miRNA signatures may be potential candidates for the clinical diagnosis, prognosis and treatment of myeloid leukemias.

  17. Cortical network from human embryonic stem cells

    OpenAIRE

    2010-01-01

    Abstract The connection of embryonic stem cell technology and developmental biology provides valuable tools to decipher the mechanisms underlying human brain development and diseases, especially among neuronal populations, that are not readily available in primary cultures. It is obviously the case of neurons forming the human cerebral cortex. In the images that are presented, the neurons were generated in vitro from human embryonic stem cells via forebrain-like progenitors. Maintained in cul...

  18. Using Proteomics to 1) Identify the Bone Marrow Homing Receptors Expressed on Human Hematopoietic Stem Cells and 2) Elucidate Critical Signaling Pathways Responsible for the Blockage of Hematopoietic Differentiation in Leukemia

    KAUST Repository

    Chin, Chee J.

    2011-05-22

    Successful hematopoiesis requires the trafficking of hematopoietic stem/progenitor cells (HSPCs) to their bone marrow (BM) niche, where they can differentiate to produce all blood lineages. Leukemia arises when there is a blockage of differentiation and uncontrolled proliferation in the hematopoietic cells during their development. To refine therapies for leukemia, this study sought to improve the homing of healthy donor HSPCs for better transplantation and to find new candidates for differentiating and blocking proliferation in leukemic cells. Characterizing the molecular effectors mediating cell migration forms the basis for improving clinical transplantation of HSPCs. E-selectin/ligand interactions play a critical role in the homing of HSPCs to the BM, however, the identity of E-selectin ligands remains elusive. We aimed to use mass spectrometry (MS) to fully analyze the E-selectin ligands expressed on HSPCs. Immunoprecipitation studies coupled with MS confirmed the expression of three known E-selectin ligands, the hematopoietic cell E-/L-selectin ligand (HCELL), P-selectin glycoprotein ligand-1 (PSGL-1) and CD43, and revealed the presence of many interesting candidates on HSPCs-like cell line and on primary human BM CD34+ cells. The MS dataset represents a rich resource for further characterization of E-selectin ligands, which will lead to improvement of HSPCs transplantation. 4 Understanding the critical pathways underlying the initiation and maintenance of leukemia plays a key role in treating acute myeloid leukemia (AML). Ligation of the glycoprotein, CD44, using monoclonal antibodies or its natural ligand, hyaluronic acid, drives the differentiation of immature leukemic cells towards mature terminally differentiated cells, inhibits their proliferation and in some case induces their apoptosis. The aim of this study is to characterize the phosphoproteome of AML cells in response to CD44-induced differentiation. This will afford novel insights into the

  19. Clinical and biological impact of TET2 mutations and expression in younger adult AML patients treated within the EORTC/GIMEMA AML-12 clinical trial

    NARCIS (Netherlands)

    Aslanyan, M.G.; Kroeze, L.; Langemeijer, S.M.C.; Koorenhof-Scheele, T.N.; Massop, M.; Hoogen, P. van; Linders, E.H.P.; Locht, L.T.F. van de; Tonnissen, E.; Heijden, A. van der; Silva-Coelho, P.; Cilloni, D.; Saglio, G.; Marie, J.P.; Tang, R.; Labar, B.; Amadori, S.; Muus, P.; Willemze, R.; Marijt, E.W.; Witte, T.J. de; Reijden, B.A. van der; Suciu, S.; Jansen, J.H.

    2014-01-01

    We assessed the prognostic impact of TET2 mutations and mRNA expression in a prospective cohort of 357 adult AML patients < 60 years of age enrolled in the European Organization For Research and Treatment of Cancer (EORTC)/Gruppo Italiano Malattie Ematologiche dell' Adulto (GIMEMA) AML-12 06991 c

  20. Transcriptome Profiling of Pediatric Core Binding Factor AML.

    Directory of Open Access Journals (Sweden)

    Chih-Hao Hsu

    Full Text Available The t(8;21 and Inv(16 translocations disrupt the normal function of core binding factors alpha (CBFA and beta (CBFB, respectively. These translocations represent two of the most common genomic abnormalities in acute myeloid leukemia (AML patients, occurring in approximately 25% pediatric and 15% of adult with this malignancy. Both translocations are associated with favorable clinical outcomes after intensive chemotherapy, and given the perceived mechanistic similarities, patients with these translocations are frequently referred to as having CBF-AML. It remains uncertain as to whether, collectively, these translocations are mechanistically the same or impact different pathways in subtle ways that have both biological and clinical significance. Therefore, we used transcriptome sequencing (RNA-seq to investigate the similarities and differences in genes and pathways between these subtypes of pediatric AMLs. Diagnostic RNA from patients with t(8;21 (N = 17, Inv(16 (N = 14, and normal karyotype (NK, N = 33 were subjected to RNA-seq. Analyses compared the transcriptomes across these three cytogenetic subtypes, using the NK cohort as the control. A total of 1291 genes in t(8;21 and 474 genes in Inv(16 were differentially expressed relative to the NK controls, with 198 genes differentially expressed in both subtypes. The majority of these genes (175/198; binomial test p-value < 10(-30 are consistent in expression changes among the two subtypes suggesting the expression profiles are more similar between the CBF cohorts than in the NK cohort. Our analysis also revealed alternative splicing events (ASEs differentially expressed across subtypes, with 337 t(8;21-specific and 407 Inv(16-specific ASEs detected, the majority of which were acetylated proteins (p = 1.5 x 10(-51 and p = 1.8 x 10(-54 for the two subsets. In addition to known fusions, we identified and verified 16 de novo fusions in 43 patients, including three fusions involving NUP98 in six

  1. Results of the AIEOP AML 2002/01 multicenter prospective trial for the treatment of children with acute myeloid leukemia.

    Science.gov (United States)

    Pession, Andrea; Masetti, Riccardo; Rizzari, Carmelo; Putti, Maria Caterina; Casale, Fiorina; Fagioli, Franca; Luciani, Matteo; Lo Nigro, Luca; Menna, Giuseppe; Micalizzi, Concetta; Santoro, Nicola; Testi, Anna Maria; Zecca, Marco; Biondi, Andrea; Pigazzi, Martina; Rutella, Sergio; Rondelli, Roberto; Basso, Giuseppe; Locatelli, Franco

    2013-07-11

    We evaluated the outcome of 482 children with acute myeloid leukemia (AML) enrolled in the Associazione Italiana di Ematologia e Oncologia Pediatrica AML 2002/01 trial. Treatment was stratified according to risk group; hematopoietic stem cell transplantation (HSCT) was used in high-risk (HR) children. Patients with core binding factor leukemia achieving complete remission (CR) after the first induction course were considered standard risk (SR; 99 patients), whereas the others (n = 383) were assigned to the HR group. Allogeneic (ALLO) or autologous (AUTO) HSCT was employed, respectively, in 141 and 102 HR patients after consolidation therapy. CR, early death, and induction failure rates were 87%, 3%, and 10%, respectively. Relapse occurred in 24% of patients achieving CR. The 8-year overall survival (OS), event-free survival (EFS), and disease-free survival (DFS) were 68%, 55%, and 63%, respectively. OS, EFS, and DFS for SR and HR patients were 83%, 63%, and 66% and 64%, 53%, and 62%. DFS was 63% and 73% for HR patients given AUTO-HSCT and ALLO-HSCT, respectively. In multivariate analysis, risk group, white blood cell >100 × 10(9)/L at diagnosis, and monosomal karyotype predicted poorer EFS. Risk-oriented treatment and broad use of HSCT result in a long-term EFS comparing favorably with previously published studies on childhood AML.

  2. Human hematopoietic cell culture, transduction, and analyses

    DEFF Research Database (Denmark)

    Bonde, Jesper; Wirthlin, Louisa; Kohn, Donald B;

    2008-01-01

    This unit provides methods for introducing genes into human hematopoietic progenitor cells. The Basic Protocol describes isolation of CD34(+) cells, transduction of these cells with a retroviral vector on fibronectin-coated plates, assaying the efficiency of transduction, and establishing long...

  3. Human embryonic stem cells derived by somatic cell nuclear transfer.

    Science.gov (United States)

    Tachibana, Masahito; Amato, Paula; Sparman, Michelle; Gutierrez, Nuria Marti; Tippner-Hedges, Rebecca; Ma, Hong; Kang, Eunju; Fulati, Alimujiang; Lee, Hyo-Sang; Sritanaudomchai, Hathaitip; Masterson, Keith; Larson, Janine; Eaton, Deborah; Sadler-Fredd, Karen; Battaglia, David; Lee, David; Wu, Diana; Jensen, Jeffrey; Patton, Phillip; Gokhale, Sumita; Stouffer, Richard L; Wolf, Don; Mitalipov, Shoukhrat

    2013-06-06

    Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.

  4. Human Neuroepithelial Cells Express NMDA Receptors

    Directory of Open Access Journals (Sweden)

    Cappell B

    2003-11-01

    Full Text Available Abstract L-glutamate, an excitatory neurotransmitter, binds to both ionotropic and metabotropic glutamate receptors. In certain parts of the brain the BBB contains two normally impermeable barriers: 1 cerebral endothelial barrier and 2 cerebral epithelial barrier. Human cerebral endothelial cells express NMDA receptors; however, to date, human cerebral epithelial cells (neuroepithelial cells have not been shown to express NMDA receptor message or protein. In this study, human hypothalamic sections were examined for NMDA receptors (NMDAR expression via immunohistochemistry and murine neuroepithelial cell line (V1 were examined for NMDAR via RT-PCR and Western analysis. We found that human cerebral epithelium express protein and cultured mouse neuroepithelial cells express both mRNA and protein for the NMDA receptor. These findings may have important consequences for neuroepithelial responses during excitotoxicity and in disease.

  5. 实时定量荧光反转录-聚合酶链反应检测急性白血病AML1/ETO融合基因%Real-time RT-PCR for detection and quantification of AML1/ETO leukemia fusion gene

    Institute of Scientific and Technical Information of China (English)

    李斌; 喻镁佳; 梁洋; 李晓进; 胡杰; 陈琪; 李惠民

    2009-01-01

    Objective To set up real-time quantitative RT-PCR technique and measure leukemia fusion gene transcripts in patients with AML of FAB-M_2 subtype,and also to investigate the positive rate in patients and the relationship between the AMLI/ETO mRNA levels and the response rate after chemical therapy.Methods The plasmid containing the AMLI/ETO fusion gene sequences were constructed from myeloid cell lines Kasumi-1 (expressing AML1/ETO)to establish the standard curves,A TaqMan based realtime quantitative RT-PCR was performed to measure aberrant fusion gene transcripts in 45 samples of peripheral blood(PB) or bone marrow (BM) from 25 newly diagnosed patients with AML-M_2.All these 25 patients were diagnosed by the FCM(flow cytometry)and bone marrow molecular cytogenetics,and received the induce remission therapy with MA (Mitoxantrone+Ara-C).Results As a result,the AML1/ETO fusion gene transcripts were detected in 7(28%)out of 25 AML-M_2 patients (the ratios of AML1/ETO/ABL vary from 0.01 to 19.2),in which 5 patients were found t(8;21)(q22;q22).The transcript level of AML1/ETO fusion gene varied from the clinical situation of patients.These 7 patients with AML1/ETO fusion gene got complete remission(CR) after the first MA therapy,and the fusion gene reduced by 3 log in AML1/ETO/ABL.Only 11 patients got CR in 18 patients without AML1/ETO fusion gene.By following up these 7 patients with AML1/ ETO fusion gene kept persistent CR for 6 months.Conclusion It was concluded that real-time quantitative PCR is a reliable,innovative and promising technology with high sensitivity and speciality.It has potential clinical value for diagnosis,tumor typing,treatment selection,measuring the tumor load,monitoring fusion gene expression level and evaluating therapeutic strategy.It is worthy to apply in the clinical practice.%目的 建立实时定量荧光反转录-聚合酶链反应(RQ RT-PCR)的方法并用来检测急性髓系白血病(AML)-M_2:患者中 AML1/ETO融合基因的拷贝

  6. Tumor necrosis factor downregulates granulocyte-colony-stimulating factor receptor expression on human acute myeloid leukemia cells and granulocytes.

    OpenAIRE

    Elbaz, O; Budel, L M; Hoogerbrugge, H; Touw, I P; Delwel, R.; Mahmoud, L A; Löwenberg, B. (Bernward)

    1991-01-01

    Tumor necrosis factor (TNF) inhibits granulocyte-colony-stimulating factor (G-CSF)-induced human acute myeloid leukemia (AML) growth in vitro. Incubation of blasts from three patients with AML in serum-free medium with TNF (10(3) U/ml), and subsequent binding studies using 125I-G-CSF reveal that TNF downregulates the numbers of G-CSF receptors by approximately 70%. G-CSF receptor numbers on purified blood granulocytes are also downmodulated by TNF. Downregulation of G-CSF receptor expression ...

  7. Long term maintenance of myeloid leukemic stem cells cultured with unrelated human mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Sawa Ito

    2015-01-01

    Full Text Available Mesenchymal stromal cells (MSCs support the growth and differentiation of normal hematopoietic stem cells (HSCs. Here we studied the ability of MSCs to support the growth and survival of leukemic stem cells (LSCs in vitro. Primary leukemic blasts isolated from the peripheral blood of 8 patients with acute myeloid leukemia (AML were co-cultured with equal numbers of irradiated MSCs derived from unrelated donor bone marrow, with or without cytokines for up to 6 weeks. Four samples showed CD34+CD38− predominance, and four were predominantly CD34+CD38+. CD34+ CD38− predominant leukemia cells maintained the CD34+ CD38− phenotype and were viable for 6 weeks when co-cultured with MSCs compared to co-cultures with cytokines or medium only, which showed rapid differentiation and loss of the LSC phenotype. In contrast, CD34+ CD38+ predominant leukemic cells maintained the CD34+CD38+ phenotype when co-cultured with MSCs alone, but no culture conditions supported survival beyond 4 weeks. Cell cycle analysis showed that MSCs maintained a higher proportion of CD34+ blasts in G0 than leukemic cells cultured with cytokines. AML blasts maintained in culture with MSCs for up to 6 weeks engrafted NSG mice with the same efficiency as their non-cultured counterparts, and the original karyotype persisted after co-culture. Chemosensitivity and transwell assays suggest that MSCs provide pro-survival benefits to leukemic blasts through cell–cell contact. We conclude that MSCs support long-term maintenance of LSCs in vitro. This simple and inexpensive approach will facilitate basic investigation of LSCs and enable screening of novel therapeutic agents targeting LSCs.

  8. Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor

    Science.gov (United States)

    Parks, Kelsey

    2009-01-01

    Space flight has been shown to alter the astronauts immune systems. Because immune performance is complex and reflects the influence of multiple organ systems within the host, scientists sought to understand the potential impact of microgravity alone on the cellular mechanisms critical to immunity. Lymphocytes and their differentiated immature form, lymphoblasts, play an important and integral role in the body's defense system. T cells, one of the three major types of lymphocytes, play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors. Reported studies have shown that spaceflight can affect the expression of cell surface markers. Cell surface markers play an important role in the ability of cells to interact and to pass signals between different cells of the same phenotype and cells of different phenotypes. Recent evidence suggests that cell-cycle regulators are essential for T-cell function. To trigger an effective immune response, lymphocytes must proliferate. The objective of this project is to investigate the changes in growth of human cells cultured in rotating bioreactors and to measure the growth rate and the cell cycle distribution for different human cell types. Human lymphocytes and lymphoblasts will be cultured in a bioreactor to simulate aspects of microgravity. The bioreactor is a cylindrical culture vessel that incorporates the aspects of clinostatic rotation of a solid fluid body around a horizontal axis at a constant speed, and compensates gravity by rotation and places cells within the fluid body into a sustained free-fall. Cell cycle progression and cell proliferation of the lymphocytes will be measured for a number of days. In addition, RNA from the cells will be isolated for expression of genes related in cell cycle regulations.

  9. Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor

    Science.gov (United States)

    Parks, Kelsey

    2009-01-01

    Space flight has been shown to alter the astronauts immune systems. Because immune performance is complex and reflects the influence of multiple organ systems within the host, scientists sought to understand the potential impact of microgravity alone on the cellular mechanisms critical to immunity. Lymphocytes and their differentiated immature form, lymphoblasts, play an important and integral role in the body's defense system. T cells, one of the three major types of lymphocytes, play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors. Reported studies have shown that spaceflight can affect the expression of cell surface markers. Cell surface markers play an important role in the ability of cells to interact and to pass signals between different cells of the same phenotype and cells of different phenotypes. Recent evidence suggests that cell-cycle regulators are essential for T-cell function. To trigger an effective immune response, lymphocytes must proliferate. The objective of this project is to investigate the changes in growth of human cells cultured in rotating bioreactors and to measure the growth rate and the cell cycle distribution for different human cell types. Human lymphocytes and lymphoblasts will be cultured in a bioreactor to simulate aspects of microgravity. The bioreactor is a cylindrical culture vessel that incorporates the aspects of clinostatic rotation of a solid fluid body around a horizontal axis at a constant speed, and compensates gravity by rotation and places cells within the fluid body into a sustained free-fall. Cell cycle progression and cell proliferation of the lymphocytes will be measured for a number of days. In addition, RNA from the cells will be isolated for expression of genes related in cell cycle regulations.

  10. [Clinical efficacy of decitabine combined with modified CAG regimen for relapsed-refractory acute myeloid leukemia with AML1-ETO⁺].

    Science.gov (United States)

    Jing, Yu; Zhu, Cheng-Ying; Zhang, Qi; Niu, Jian-Hua; Yang, Hua; Liu, Shi-Yan; Zhu, Hai-Yan; Yu, Li

    2014-10-01

    This study was aimed to investigate the clinical characteristics of relapsed-refractory acute myeloid leukemia (AML) with AML1-ETO⁺, and its therapeutic efficacy and side effects when decitabine combined with modified CAG regimen was used. Clinical data of 5 cases of AML with AML1-ETO⁺ from January 2013 to Agust 2013 were analyzed retrospectively. The analyzed data included age, sex, initial symptoms, peripheral blood and bone marrow characteristics. Meanwhile, the therapeutic effecacy and side effects of decitabine combined with modified CAG regimen were evaluated. The 5 patients were with median age of 35 (17-43) years. Among these 5 patients, 2 patients were relapsed and other 3 patients were relapsed-refractory patients, their median white blood cell count was 12.55 (7.8-66.55) × 10⁹/L, median platelets count was 44 (20-72) × 10⁹/L, median hemoglobin level was 110 (77-128) g/L, median lactate dehydrogenase level was 312.9 U/L (123.6-877.8) at the initial diagnosis. The results showed that after decitabine combined with modified CAG regimen was administered, 4 patients achieved complete remission, 1 patient did not achieve remission, the overall remission rate was 80% (4/5). The main side effects of this regimen was myelosuppression, these were no new lung infection and other serious complications, one case without complete remission treated with FLAG once again died of heart failure when being mobilized for transplantation. It is concluded that according to preliminary results of decitabine combined with modified CAG regimen for relapsed and refractory AML patients with AML1-ETO⁺ displays higher remission rate and lower side effects, which worthy to further explore for clinal application.

  11. P-glycoprotein and breast cancer resistance protein in acute myeloid leukaemia cells treated with the Aurora-B Kinase Inhibitor barasertib-hQPA

    Directory of Open Access Journals (Sweden)

    Russell Nigel H

    2011-06-01

    Full Text Available Abstract Background Aurora kinases play an essential role in orchestrating chromosome alignment, segregation and cytokinesis during mitotic progression, with both aurora-A and B frequently over-expressed in a variety of human malignancies. Over-expression of the ABC drug transporter proteins P-glycoprotein (Pgp and Breast cancer resistance protein (BCRP is a major obstacle for chemotherapy in many tumour types with Pgp conferring particularly poor prognosis in acute myeloid leukaemia (AML. Barasertib-hQPA is a highly selective inhibitor of aurora-B kinase that has shown tumouricidal activity against a range tumour cell lines including those of leukaemic AML origin. Methods Effect of barasertib-hQPA on the pHH3 biomarker and cell viability was measured in a panel of leukaemic cell lines and 37 primary AML samples by flow cytometry. Pgp status was determined by flow cytometry and BCRP status by flow cytometry and real-time PCR. Results In this study we report the creation of the cell line OCI-AML3DNR, which over-expresses Pgp but not BCRP or multidrug resistance-associated protein (MRP, through prolonged treatment of OCI-AML3 cells with daunorubicin. We demonstrate that Pgp (OCI-AML3DNR and KG-1a and BCRP (OCI-AML6.2 expressing AML cell lines are less sensitive to barasertib-hQPA induced pHH3 inhibition and subsequent loss of viability compared to transporter negative cell lines. We also show that barasertib-hQPA resistance in these cell lines can be reversed using known Pgp and BCRP inhibitors. We report that barasertib-hQPA is not an inhibitor of Pgp or BCRP, but by using 14[C]-barasertib-hQPA that it is effluxed by these transporters. Using phosphoHistone H3 (pHH3 as a biomarker of barasertib-hQPA responsiveness in primary AML blasts we determined that Pgp and BCRP positive primary samples were less sensitive to barasertib-hQPA induced pHH3 inhibition (p = 50 inhibition of pHH3 by barasertib-hQPA was achieved in 94.6% of these samples after 1

  12. Spontaneous Remission in an Older Patient with Relapsed FLT3 ITD Mutant AML

    Directory of Open Access Journals (Sweden)

    Pankit Vachhani

    2016-01-01

    Full Text Available Spontaneous remission (SR of acute myeloid leukemia (AML is a very rare phenomenon. AML characterized by FLT3 internal tandem duplication (FLT3 ITD is typically associated with an aggressive clinical course with rapid progression, relapse, and short overall survival in the absence of transplantation. We report here the first case of SR of FLT3 ITD mutant AML in the literature. Our patient was an elderly woman with relapsed NPM1 and FLT3 ITD mutant AML whose disease underwent SR for a brief duration without precipitating cause. We review the potential immune mechanisms underlying SR in AML and discuss the implications for novel immunotherapeutic approaches for FLT3 mutant AML.

  13. Derivation of naive human embryonic stem cells.

    Science.gov (United States)

    Ware, Carol B; Nelson, Angelique M; Mecham, Brigham; Hesson, Jennifer; Zhou, Wenyu; Jonlin, Erica C; Jimenez-Caliani, Antonio J; Deng, Xinxian; Cavanaugh, Christopher; Cook, Savannah; Tesar, Paul J; Okada, Jeffrey; Margaretha, Lilyana; Sperber, Henrik; Choi, Michael; Blau, C Anthony; Treuting, Piper M; Hawkins, R David; Cirulli, Vincenzo; Ruohola-Baker, Hannele

    2014-03-25

    The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes, and forced expression of OCT4, KLF4, and KLF2 allows maintenance of human cells in a naïve state [Hanna J, et al. (2010) Proc Natl Acad Sci USA 107(20):9222-9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible.

  14. PRL-3, a metastasis associated tyrosine phosphatase, is involved in FLT3-ITD signaling and implicated in anti-AML therapy.

    Directory of Open Access Journals (Sweden)

    Jianbiao Zhou

    Full Text Available Combination with other small molecule drugs represents a promising strategy to improve therapeutic efficacy of FLT3 inhibitors in the clinic. We demonstrated that combining ABT-869, a FLT3 inhibitor, with SAHA, a HDAC inhibitor, led to synergistic killing of the AML cells with FLT3 mutations and suppression of colony formation. We identified a core gene signature that is uniquely induced by the combination treatment in 2 different leukemia cell lines. Among these, we showed that downregulation of PTP4A3 (PRL-3 played a role in this synergism. PRL-3 is downstream of FLT3 signaling and ectopic expression of PRL-3 conferred therapeutic resistance through upregulation of STAT (signal transducers and activators of transcription pathway activity and anti-apoptotic Mcl-1 protein. PRL-3 interacts with HDAC4 and SAHA downregulates PRL-3 via a proteasome dependent pathway. In addition, PRL-3 protein was identified in 47% of AML cases, but was absent in myeloid cells in normal bone marrows. Our results suggest such combination therapies may significantly improve the therapeutic efficacy of FLT3 inhibitors. PRL-3 plays a potential pathological role in AML and it might be a useful therapeutic target in AML, and warrant clinical investigation.

  15. PRL-3, a metastasis associated tyrosine phosphatase, is involved in FLT3-ITD signaling and implicated in anti-AML therapy.

    Science.gov (United States)

    Zhou, Jianbiao; Bi, Chonglei; Chng, Wee-Joo; Cheong, Lip-Lee; Liu, Shaw-Cheng; Mahara, Sylvia; Tay, Kian-Ghee; Zeng, Qi; Li, Jie; Guo, Ke; Tan, Cheng Peow Bobby; Yu, Hanry; Albert, Daniel H; Chen, Chien-Shing

    2011-05-12

    Combination with other small molecule drugs represents a promising strategy to improve therapeutic efficacy of FLT3 inhibitors in the clinic. We demonstrated that combining ABT-869, a FLT3 inhibitor, with SAHA, a HDAC inhibitor, led to synergistic killing of the AML cells with FLT3 mutations and suppression of colony formation. We identified a core gene signature that is uniquely induced by the combination treatment in 2 different leukemia cell lines. Among these, we showed that downregulation of PTP4A3 (PRL-3) played a role in this synergism. PRL-3 is downstream of FLT3 signaling and ectopic expression of PRL-3 conferred therapeutic resistance through upregulation of STAT (signal transducers and activators of transcription) pathway activity and anti-apoptotic Mcl-1 protein. PRL-3 interacts with HDAC4 and SAHA downregulates PRL-3 via a proteasome dependent pathway. In addition, PRL-3 protein was identified in 47% of AML cases, but was absent in myeloid cells in normal bone marrows. Our results suggest such combination therapies may significantly improve the therapeutic efficacy of FLT3 inhibitors. PRL-3 plays a potential pathological role in AML and it might be a useful therapeutic target in AML, and warrant clinical investigation.

  16. Cytosine arabinoside induces costimulatory molecule expression in acute myeloid leukemia cells.

    Science.gov (United States)

    Vereecque, R; Saudemont, A; Quesnel, B

    2004-07-01

    Chemotherapeutic drugs kill cancer cells mainly by direct cytotoxicity, but they might also induce a stronger host immune response by causing the tumor to produce costimulatory cell surface molecules like CD80. We previously reported that in myeloid leukemic cells, gamma-irradiation induced CD80 expression. In this study, we show that cytosine arabinoside (Ara-C), even at low doses, induced CD80 expression in vitro in mouse DA1-3b leukemic cells, by a mechanism that involved reactive oxygen species. In vivo experiments in the mouse DA1-3b/C3H whole-animal acute myeloid leukemia (AML) model showed that injection of Ara-C induced expression of CD80 and CD86, and decreased expression of B7-H1, indicating that chemotherapy can modify costimulatory molecule expression in vivo, in a way not necessarily observed in vitro. Mouse leukemic cells exposed in vivo to Ara-C were more susceptible to specific cytotoxic lymphocyte (CTL)-mediated killing. Ara-C also induced CD80 or CD86 expression in 14 of 21 primary cultured human AML samples. In humans being treated for AML, induction chemotherapy increased CD86 expression in the leukemic cells. These findings indicate possible synergistic strategies between CTL-based immunotherapy and chemotherapy for treatment. They also suggest an additional mechanism by which chemotherapy can eradicate AML blasts.

  17. Regulatory T Cells in Human Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Dong-Jun Peng

    2012-01-01

    Full Text Available Multiple layers of suppressive components including regulatory T (TReg cells, suppressive antigen-presenting cells, and inhibitory cytokines form suppressive networks in the ovarian cancer microenvironment. It has been demonstrated that as a major suppressive element, TReg cells infiltrate tumor, interact with several types of immune cells, and mediate immune suppression through different molecular and cellular mechanisms. In this paper, we focus on human ovarian cancer and will discuss the nature of TReg cells including their subsets, trafficking, expansion, and function. We will briefly review the development of manipulation of TReg cells in preclinical and clinical settings.

  18. Generation of pancreatic islet cells from human embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG DongHui; JIANG Wei; SHI Yan; DENG HongKui

    2009-01-01

    Efficiently obtaining functional pancreaUc islet cells derived from human embryonic stem (hES) cells not only provides great potential to solve the shortage of islets sources for type I diabetes cell therapy,but also benefits the study of the development of the human pancreas and diabetes pathology. In 2001,hES cells were reported to have the capacity to generate insulin-producing cells by spontaneous differentiation in vitro. Since then, many strategies (such as overexpression of key transcription factors,delivery of key proteins for pancreatic development, co-transplantation of differentiated hES cells along with fetal pancreas, stepwise differentiation by mimicking in vivo pancreatic development) have been employed in order to induce the differentiation of pancreatic islet cells from hES cells. Moreover, patient-specific induced pluripotent stem (iPS) cells can be generated by reprogramming somatic cells.iPS cells have characteristics similar to those of ES cells and offer a new cell source for type I diabetes cell therapy that reduces the risk of immunologic rejection. In this review, we summarize the recent progress made in the differentiation of hES and iPS cells into functional pancreatic islet cells and discuss the challenges for their future study.

  19. Generation of pancreatic islet cells from human embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Efficiently obtaining functional pancreatic islet cells derived from human embryonic stem(hES) cells not only provides great potential to solve the shortage of islets sources for type I diabetes cell therapy,but also benefits the study of the development of the human pancreas and diabetes pathology.In 2001,hES cells were reported to have the capacity to generate insulin-producing cells by spontaneous differentiation in vitro.Since then,many strategies(such as overexpression of key transcription factors,delivery of key proteins for pancreatic development,co-transplantation of differentiated hES cells along with fetal pancreas,stepwise differentiation by mimicking in vivo pancreatic development) have been employed in order to induce the differentiation of pancreatic islet cells from hES cells.Moreover,patient-specific induced pluripotent stem(iPS) cells can be generated by reprogramming somatic cells.iPS cells have characteristics similar to those of ES cells and offer a new cell source for type I diabetes cell therapy that reduces the risk of immunologic rejection.In this review,we summarize the recent progress made in the differentiation of hES and iPS cells into functional pancreatic islet cells and discuss the challenges for their future study.

  20. Plasma membrane proteomics of human embryonic stem cells and human embryonal carcinoma cells.

    NARCIS (Netherlands)

    Dormeyer, W.; van Hoof, D.; Braam, S.R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.

    2008-01-01

    Human embryonic stem cells (hESCs) are of immense interest in regenerative medicine as they can self-renew indefinitely and can give rise to any adult cell type. Human embryonal carcinoma cells (hECCs) are the malignant counterparts of hESCs found in testis tumors. hESCs that have acquired chromosom

  1. The role of therapeutic leukapheresis in hyperleukocytotic AML.

    Directory of Open Access Journals (Sweden)

    Friederike Pastore

    Full Text Available PURPOSE: Hyperleukocytosis in AML with leukostasis is a serious life-threatening condition leading to a high early mortality which requires immediate cytoreductive therapy. Therapeutic leukapheresis is currently recommended by the American Society of Apheresis in patients with a WBC>100 G/l with signs of leukostasis, but the role of prophylactic leukapheresis before clinical signs of leukostasis occur is unclear. PATIENTS: We retrospectively analyzed the role of leukapheresis in 52 patients (median age 60 years with hyperleukocytotic AML with and without clinical signs of leukostasis. Since leukapheresis was performed more frequently in patients with signs of leukostasis due to the therapeutic policy in our hospital, we developed a risk score for early death within seven days after start of therapy (EDd7 to account for this selection bias and to independently measure the effect of leukapheresis on EDd7. RESULTS: 20 patients received leukapheresis in combination to chemotherapy compared to 32 patients who received chemotherapy only. In a multivariate logistic regression model for the estimation of the probability of EDd7 thromboplastin time and creatinine remained as independent significant parameters and were combined to create an EDd7 risk score. The effect of leukapheresis on EDd7 was evaluated in a bivariate logistic regression together with the risk score. Leukapheresis did not significantly change early mortality in all patients with a WBC≥100 G/l. DISCUSSION: Prophylactic leukapheresis in hyperleukocytotic patients with and without leukostasis did not improve early mortality in our retrospective study. Larger and prospective clinical trials are needed to validate the risk score and to further explore the role of leukapheresis in AML with hyperleukocytosis.

  2. Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

    Science.gov (United States)

    Unger, Christian; Felldin, Ulrika; Rodin, Sergey; Nordenskjöld, Agneta; Dilber, Sirac; Hovatta, Outi

    2016-02-03

    After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts.

  3. Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Varga, Nora [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Vereb, Zoltan; Rajnavoelgyi, Eva [Department of Immunology, Medical and Health Science Centre, University of Debrecen, Debrecen (Hungary); Nemet, Katalin; Uher, Ferenc; Sarkadi, Balazs [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Apati, Agota, E-mail: apati@kkk.org.hu [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary)

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.

  4. Outcome of poor response Paediatric AML using early SCT

    DEFF Research Database (Denmark)

    Wareham, Neval E; Heilmann, Carsten; Abrahamsson, Jonas;

    2013-01-01

    treated with SCT. MATERIAL AND METHODS: Treatment was given according to the NOPHO-AML 2004 protocol. All patients received AIET (Cytarabine, Idarubicin, Etoposide, Thioguanine) and AM (Cytarabine, Mitoxantrone) as induction. We included poor response defined as > 15% blasts on day 15 after AIET (n = 17......) or > 5% blasts after AM (n = 14, refractory disease). Poor response patients received intensively timed induction and proceeded to SCT when a donor was available. RESULTS: Thirty-one of 267 evaluable patients (12%) had a poor response. SCT was performed in 25; using matched unrelated donors in 13...

  5. Azacitidine for Front-Line Therapy of Patients with AML: Reproducible Efficacy Established by Direct Comparison of International Phase 3 Trial Data with Registry Data from the Austrian Azacitidine Registry of the AGMT Study Group

    Directory of Open Access Journals (Sweden)

    Lisa Pleyer

    2017-02-01

    Full Text Available We recently published a clinically-meaningful improvement in median overall survival (OS for patients with acute myeloid leukaemia (AML, >30% bone marrow (BM blasts and white blood cell (WBC count ≤15 G/L, treated with front-line azacitidine versus conventional care regimens within a phase 3 clinical trial (AZA-AML-001; NCT01074047; registered: February 2010. As results obtained in clinical trials are facing increased pressure to be confirmed by real-world data, we aimed to test whether data obtained in the AZA-AML-001 trial accurately represent observations made in routine clinical practice by analysing additional AML patients treated with azacitidine front-line within the Austrian Azacitidine Registry (AAR; NCT01595295; registered: May 2012 and directly comparing patient-level data of both cohorts. We assessed the efficacy of front-line azacitidine in a total of 407 patients with newly-diagnosed AML. Firstly, we compared data from AML patients with WBC ≤ 15 G/L and >30% BM blasts included within the AZA-AML-001 trial treated with azacitidine (“AML-001” cohort; n = 214 with AAR patients meeting the same inclusion criteria (“AAR (001-like” cohort; n = 95. The current analysis thus represents a new sub-analysis of the AML-001 trial, which is directly compared with a new sub-analysis of the AAR. Baseline characteristics, azacitidine application, response rates and OS were comparable between all patient cohorts within the trial or registry setting. Median OS was 9.9 versus 10.8 months (p = 0.616 for “AML-001” versus “AAR (001-like” cohorts, respectively. Secondly, we pooled data from both cohorts (n = 309 and assessed the outcome. Median OS of the pooled cohorts was 10.3 (95% confidence interval: 8.7, 12.6 months, and the one-year survival rate was 45.8%. Thirdly, we compared data from AAR patients meeting AZA-AML-001 trial inclusion criteria (n = 95 versus all AAR patients with World Health Organization (WHO-defined AML (

  6. Stem cells in the human breast

    DEFF Research Database (Denmark)

    Petersen, Ole William; Polyak, Kornelia

    2010-01-01

    The origins of the epithelial cells participating in the development, tissue homeostasis, and cancer of the human breast are poorly understood. However, emerging evidence suggests a role for adult tissue-specific stem cells in these processes. In a hierarchical manner, these generate the two main...

  7. Human hair genealogies and stem cell latency

    Directory of Open Access Journals (Sweden)

    Tavaré Simon

    2006-02-01

    Full Text Available Abstract Background Stem cells divide to reproduce themselves and produce differentiated progeny. A fundamental problem in human biology has been the inability to measure how often stem cells divide. Although it is impossible to observe every division directly, one method for counting divisions is to count replication errors; the greater the number of divisions, the greater the numbers of errors. Stem cells with more divisions should produce progeny with more replication errors. Methods To test this approach, epigenetic errors (methylation in CpG-rich molecular clocks were measured from human hairs. Hairs exhibit growth and replacement cycles and "new" hairs physically reappear even on "old" heads. Errors may accumulate in long-lived stem cells, or in their differentiated progeny that are eventually shed. Results Average hair errors increased until two years of age, and then were constant despite decades of replacement, consistent with new hairs arising from infrequently dividing bulge stem cells. Errors were significantly more frequent in longer hairs, consistent with long-lived but eventually shed mitotic follicle cells. Conclusion Constant average hair methylation regardless of age contrasts with the age-related methylation observed in human intestine, suggesting that error accumulation and therefore stem cell latency differs among tissues. Epigenetic molecular clocks imply similar mitotic ages for hairs on young and old human heads, consistent with a restart with each new hair, and with genealogies surreptitiously written within somatic cell genomes.

  8. Therapy-related myelodysplastic syndrome/acute myeloid leukemia with del(7)(q22) in a patient with de novo AML.

    Science.gov (United States)

    Kim, Yang Gyun; Cho, Sun Young; Park, Tae Sung; Oh, Seung Hwan; Yoon, Hwi-Joong

    2011-01-01

    A 55-year-old Korean woman was initially diagnosed with acute myelomonocytic leukemia (AML). After induction chemotherapy was performed using cytarabine, idarubicin, and G-CSF, complete remission (CR) was subsequently achieved following reinduction chemotherapy using the same chemotherapeutic agents. Thirty-six months after the initial CR, an increase in immature cells (up to 12.0%) was observed in the patient's bone marrow. Because chromosome analysis revealed a karyotype of 46,XX,del(7)(q22) in all of the analyzed cells, the patient was diagnosed with therapy-related myelodysplastic syndrome (t-MDS). Although the patient subsequently received chemotherapy and G-CSF for neutropenia, t-MDS rapidly progressed after 3 months to therapy-related acute myeloid leukemia (t-AML). Although very rare, de novo AML can progress to a secondary MDS/AML with del(7q) after chemotherapy with cytarabine, idarubicin, and G-CSF. Further investigation into the role of genes located in 7q22 may provide more information about the mechanisms of leukemogenesis.

  9. RUNX1-ETO and RUNX1-EVI1 Differentially Reprogram the Chromatin Landscape in t(8;21) and t(3;21) AML.

    Science.gov (United States)

    Loke, Justin; Assi, Salam A; Imperato, Maria Rosaria; Ptasinska, Anetta; Cauchy, Pierre; Grabovska, Yura; Soria, Natalia Martinez; Raghavan, Manoj; Delwel, H Ruud; Cockerill, Peter N; Heidenreich, Olaf; Bonifer, Constanze

    2017-05-23

    Acute myeloid leukemia (AML) is a heterogeneous disease caused by mutations in transcriptional regulator genes, but how different mutant regulators shape the chromatin landscape is unclear. Here, we compared the transcriptional networks of two types of AML with chromosomal translocations of the RUNX1 locus that fuse the RUNX1 DNA-binding domain to different regulators, the t(8;21) expressing RUNX1-ETO and the t(3;21) expressing RUNX1-EVI1. Despite containing the same DNA-binding domain, the two fusion proteins display distinct binding patterns, show differences in gene expression and chromatin landscape, and are dependent on different transcription factors. RUNX1-EVI1 directs a stem cell-like transcriptional network reliant on GATA2, whereas that of RUNX1-ETO-expressing cells is more mature and depends on RUNX1. However, both types of AML are dependent on the continuous expression of the fusion proteins. Our data provide a molecular explanation for the differences in clinical prognosis for these types of AML. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  10. RUNX1-ETO and RUNX1-EVI1 Differentially Reprogram the Chromatin Landscape in t(8;21 and t(3;21 AML

    Directory of Open Access Journals (Sweden)

    Justin Loke

    2017-05-01

    Full Text Available Acute myeloid leukemia (AML is a heterogeneous disease caused by mutations in transcriptional regulator genes, but how different mutant regulators shape the chromatin landscape is unclear. Here, we compared the transcriptional networks of two types of AML with chromosomal translocations of the RUNX1 locus that fuse the RUNX1 DNA-binding domain to different regulators, the t(8;21 expressing RUNX1-ETO and the t(3;21 expressing RUNX1-EVI1. Despite containing the same DNA-binding domain, the two fusion proteins display distinct binding patterns, show differences in gene expression and chromatin landscape, and are dependent on different transcription factors. RUNX1-EVI1 directs a stem cell-like transcriptional network reliant on GATA2, whereas that of RUNX1-ETO-expressing cells is more mature and depends on RUNX1. However, both types of AML are dependent on the continuous expression of the fusion proteins. Our data provide a molecular explanation for the differences in clinical prognosis for these types of AML.

  11. Human neutrophils facilitate tumor cell transendothelial migration.

    LENUS (Irish Health Repository)

    Wu, Q D

    2012-02-03

    Tumor cell extravasation plays a key role in tumor metastasis. However, the precise mechanisms by which tumor cells migrate through normal vascular endothelium remain unclear. In this study, using an in vitro transendothelial migration model, we show that human polymorphonuclear neutrophils (PMN) assist the human breast tumor cell line MDA-MB-231 to cross the endothelial barrier. We found that tumor-conditioned medium (TCM) downregulated PMN cytocidal function, delayed PMN apoptosis, and concomitantly upregulated PMN adhesion molecule expression. These PMN treated with TCM attached to tumor cells and facilitated tumor cell migration through different endothelial monolayers. In contrast, MDA-MB-231 cells alone did not transmigrate. FACScan analysis revealed that these tumor cells expressed high levels of intercellular adhesion molecule-1 (ICAM-1) but did not express CD11a, CD11b, or CD18. Blockage of CD11b and CD18 on PMN and of ICAM-1 on MDA-MB-231 cells significantly attenuated TCM-treated, PMN-mediated tumor cell migration. These tumor cells still possessed the ability to proliferate after PMN-assisted transmigration. These results indicate that TCM-treated PMN may serve as a carrier to assist tumor cell transendothelial migration and suggest that tumor cells can exploit PMN and alter their function to facilitate their extravasation.

  12. A murine model of acute myeloid leukemia with Evi1 overexpression and autocrine stimulation by an intracellular form of GM-CSF in DA-3 cells.

    Science.gov (United States)

    Cardona, Maria E; Simonson, Oscar E; Oprea, Iulian I; Moreno, Pedro M D; Silva-Lara, Maria F; Mohamed, Abdalla J; Christensson, Birger; Gahrton, Gösta; Dilber, M Sirac; Smith, C I Edvard; Arteaga, H Jose

    2016-01-01

    The poor treatment response of acute myeloid leukemia (AML) overexpressing high-risk oncogenes such as EVI1, demands specific animal models for new treatment evaluations. Evi1 is a common site of activating integrations in murine leukemia virus (MLV)-induced AML and in retroviral and lentiviral gene-modified HCS. Still, a model of overt AML induced by Evi1 has not been generated. Cell lines from MLV-induced AML are growth factor-dependent and non-transplantable. Hence, for the leukemia maintenance in the infected animals, a growth factor source such as chronic immune response has been suggested. We have investigated whether these leukemias are transplantable if provided with growth factors. We show that the Evi1(+)DA-3 cells modified to express an intracellular form of GM-CSF, acquired growth factor independence and transplantability and caused an overt leukemia in syngeneic hosts, without increasing serum GM-CSF levels. We propose this as a general approach for modeling different forms of high-risk human AML using similar cell lines.

  13. IL-27 Promotes Proliferation of Human Leukemic Cell Lines Through the MAPK/ERK Signaling Pathway and Suppresses Sensitivity to Chemotherapeutic Drugs

    Science.gov (United States)

    Dilger, Paula; Bird, Chris; Wadhwa, Meenu

    2016-01-01

    IL-27 is a pleiotropic cytokine of the IL-6/IL-12 family with diverse biological functions. Previous in vivo studies have suggested the antitumor activities of IL-27 in animal models, whereas clinical observations indicate the link of IL-27 in tumor progression. IL-27 has recently been shown to cause inhibition of proliferation on primary leukemic cells from pediatric patients, but information on its role in human leukemic cell lines is limited. In the present study, we investigated the ability of IL-27 to regulate cell growth and survival of various human leukemic cell lines. Our results showed that in human leukemic cell lines coexpressing both IL-27R chains, IL-27Rα and gp130, IL-27 did not inhibit cell growth, but caused dose-dependent proliferation of the acute myeloid leukemic cell line, OCI-AML5, and the erythroleukemic cell lines, TF-1, UT-7, and UT-7/EPO. Consistent with this, IL-27 promoted cell survival and reduced TNF-α-induced apoptosis of the leukemic cell lines. IL-27 also decreased the responsiveness of the leukemic cells to chemotherapeutic drugs, cytarabine and daunorubicin. We observed that IL-27 induced the activation of STAT1/3 and ERK1/2 in the leukemic cells. Growth stimulation by IL-27 was suppressed by the specific MEK inhibitor, U0126, indicating that IL-27-induced cell proliferation is mainly mediated through the activation of the MAPK/ERK signaling pathway. The present study is the first demonstration of the proliferative and antichemotherapeutic properties of IL-27 in human leukemic cell lines, suggesting that IL-27 can play an unfavorable role in tumor growth and can be an important determinant in the chemoresponsiveness of certain subtypes of human leukemia. PMID:27119567

  14. Arginine deprivation using pegylated arginine deiminase has activity against primary acute myeloid leukemia cells in vivo.

    Science.gov (United States)

    Miraki-Moud, Farideh; Ghazaly, Essam; Ariza-McNaughton, Linda; Hodby, Katharine A; Clear, Andrew; Anjos-Afonso, Fernando; Liapis, Konstantinos; Grantham, Marianne; Sohrabi, Fareeda; Cavenagh, Jamie; Bomalaski, John S; Gribben, John G; Szlosarek, Peter W; Bonnet, Dominique; Taussig, David C

    2015-06-25

    The strategy of enzymatic degradation of amino acids to deprive malignant cells of important nutrients is an established component of induction therapy of acute lymphoblastic leukemia. Here we show that acute myeloid leukemia (AML) cells from most patients with AML are deficient in a critical enzyme required for arginine synthesis, argininosuccinate synthetase-1 (ASS1). Thus, these ASS1-deficient AML cells are dependent on importing extracellular arginine. We therefore investigated the effect of plasma arginine deprivation using pegylated arginine deiminase (ADI-PEG 20) against primary AMLs in a xenograft model and in vitro. ADI-PEG 20 alone induced responses in 19 of 38 AMLs in vitro and 3 of 6 AMLs in vivo, leading to caspase activation in sensitive AMLs. ADI-PEG 20-resistant AMLs showed higher relative expression of ASS1 than sensitive AMLs. This suggests that the resistant AMLs survive by producing arginine through this metabolic pathway and ASS1 expression could be used as a biomarker for response. Sensitive AMLs showed more avid uptake of arginine from the extracellular environment consistent with their auxotrophy for arginine. The combination of ADI-PEG 20 and cytarabine chemotherapy was more effective than either treatment alone resulting in responses in 6 of 6 AMLs tested in vivo. Our data show that arginine deprivation is a reasonable strategy in AML that paves the way for clinical trials.

  15. Regeneration-associated WNT Signaling Is Activated in Long-term Reconstituting AC133bright Acute Myeloid Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Alessandro Beghini

    2012-12-01

    Full Text Available Acute myeloid leukemia (AML is a genetically heterogeneous clonal disorder characterized by two molecularly distinct self-renewing leukemic stem cell (LSC populations most closely related to normal progenitors and organized as a hierarchy. A requirement for WNT/β-catenin signaling in the pathogenesis of AML has recently been suggested by a mouse model. However, its relationship to a specific molecular function promoting retention of self-renewing leukemia-initiating cells (LICs in human remains elusive. To identify transcriptional programs involved in the maintenance of a self-renewing state in LICs, we performed the expression profiling in normal (n = 10 and leukemic (n = 33 human long-term reconstituting AC133+ cells, which represent an expanded cell population in most AML patients. This study reveals the ligand-dependent WNT pathway activation in AC133bright AML cells and shows a diffuse expression and release of WNT10B, a hematopoietic stem cell regenerative-associated molecule. The establishment of a primary AC133+ AML cell culture (A46 demonstrated that leukemia cells synthesize and secrete WNT ligands, increasing the levels of dephosphorylated β-catenin in vivo. We tested the LSC functional activity in AC133+ cells and found significant levels of engraftment upon transplantation of A46 cells into irradiated Rag2-/-γc-/- mice. Owing to the link between hematopoietic regeneration and developmental signaling, we transplanted A46 cells into developing zebrafish. This system revealed the formation of ectopic structures by activating dorsal organizer markers that act downstream of the WNT pathway. In conclusion, our findings suggest that AC133bright LSCs are promoted by misappropriating homeostatic WNT programs that control hematopoietic regeneration.

  16. INTERACTIONS BETWEEN THE HUMAN GASTRIC CARCINOMA CELL AND THE HUMAN VASCULAR ENDOTHELIAL CELL

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To definite the interactions between the human gastric carcinoma cell and the human vascular endothelial cell during the establishment and maintenance of the tumor vascular system and the tumor hematogenous metastasis.Methods We prepared the conditioned mediums of each cell so as to study the effect of the conditioned medium on itself or others by MTT colorimetry. The comprehensive effect of interactions between two cells was determined by stratified transfilter co-culture or direct contact co-culture.Results The conditioned medium of human gastric carcinoma cell can stimulate the proliferation of the human vascular endothelial cell, but the CM of HVEC can inhibit the growth of HGCC. Both kinds of cells can inhibit the growth of itself. The ultimate comprehensive effect of the interactions between two kinds of cells was increase of total cell numbers.Conclusion There exist the complicated interactions between the human gastric carcinoma cell and the human vascular endothelial cell during the tumor angiogenesis and the tumor hematogenous metastasis. The ultimate comprehensive effect of the interactions is increase of total cells numbers and tumor volume.

  17. Fibronectin production by human mammary cells

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, M.R. (Univ. of California, Berkeley); Vlodavsky, I.; Smith, H.S.; Ford, R.; Becker, F.F.; Riggs, J.

    1981-01-01

    Human mammary cells were examined for the presence of the high-molecular-weight surface glycoprotein fibronectin. Early passage mammary epithelial cell and fibroblast cultures from both carcinomas and normal tissues were tested for the presence of cell-associated fibronectin by immunofluorescence microscopy and for the synthesis and secretion of fibronectin by specific immunoprecipitation of metabolically labeled protein. In vivo frozen sections of primary carcinomas and normal tissues were tested for the localization of fibronectin by immunofluorescence microscopy. In contrast to the extensive fibrillar networks of fibronectin found in the fibroblast cultures, the epithelial cell cultures from both tissue sources displayed a pattern of cell-associated fibronectin characterizd by powdery, punctate staining. However, the cultured epithelial cells, as well as the fibroblasts, secreted large quantities of fibronectin into the medium. Putative myoepithelial cells also displayed extensive fibrillar networks of fibronectin. The difference in cell-associated fibronectin distribution between the epithelial cells and the fibroblasts and putative myoepithelial cells provided a simple means of quantitating stromal and myoepithelial cell contamination of the mammary epithelial cells in culture. In vivo, normal tissues showed fibronectin primarily localized in the basement membrane surrounding the epithelial cells and in the stroma. Most primary carcinomas displayed powdery, punctate staining on the epithelial cells in addition to the fibronectin present in the surrounding stroma.

  18. Cytotoxic capacity of IL-15-stimulated cytokine-induced killer cells against human acute myeloid leukemia and rhabdomyosarcoma in humanized preclinical mouse models

    Directory of Open Access Journals (Sweden)

    Eva eRettinger

    2012-04-01

    Full Text Available Allogeneic stem cell transplantation (allo-SCT has become an important treatment modality for patients with high risk acute myeloid leukemia (AML and is also under investigation for soft tissue sarcomas. The therapeutic success is still limited by minimal residual disease (MRD status ultimately leading to patients’ relapse. Adoptive donor lymphocyte infusions (DLI based on MRD status using IL-15-expanded cytokine-induced killer (CIK cells may prevent relapse without causing graft-versus-host-disease (GvHD. To generate preclinical data we developed mouse models to study anti-leukemic- and anti-tumor-potential of CIK cells in vivo. Immunodeficient mice (NOD/SCID/IL2Rγc-, NSG were injected intravenously with human leukemic cell lines THP-1, SH-2 and with human rhabdomyosarcoma (RMS cell lines RH41 and RH30 at minimal doses required for leukemia or tumor engraftment. Mice transplanted with THP-1 or RH41 cells were randomly assigned for analysis of CIK cell treatment. Organs of mice were analyzed by flow cytometry as well as quantitative polymerase chain reaction (qPCR for engraftment of malignant cells and CIK cells. Potential of CIK cells to induce GvHD was determined by histological analysis. Tissues of the highest degree of THP-1 cell expansion included bone marrow (BM followed by liver, lung, spleen, peripheral blood (PB, and brain. RH30 and RH41 engraftment mainly took place in liver and lung, but was also detectable in spleen and PB. In spite of delayed CIK cell expansion compared with malignant cells, CIK cells injected at an effector to target cell (E:T ratio of 1:1 were sufficient for significant reduction of RH41 cells, whereas against fast-expanding THP-1 cells an E:T ratio of 250:1 was needed to achieve comparable results. Our preclinical in vivo mouse models showed a reliably 100% engraftment of malignant cells which is essential for analysis of anti-cancer therapy. Furthermore our data demonstrated that IL-15-activated CIK cells

  19. Laser printing of skin cells and human stem cells.

    Science.gov (United States)

    Koch, Lothar; Kuhn, Stefanie; Sorg, Heiko; Gruene, Martin; Schlie, Sabrina; Gaebel, Ralf; Polchow, Bianca; Reimers, Kerstin; Stoelting, Stephanie; Ma, Nan; Vogt, Peter M; Steinhoff, Gustav; Chichkov, Boris

    2010-10-01

    Laser printing based on laser-induced forward transfer (LIFT) is a new biofabrication technique for the arrangement of biological materials or living cells in well-defined patterns. In the current study, skin cell lines (fibroblasts/keratinocytes) and human mesenchymal stem cells (hMSC) were chosen for laser printing experiments due to their high potential in regeneration of human skin and new application possibilities of stem cell therapy. To evaluate the influence of LIFT on the cells, their survival rate, their proliferation and apoptotic activity, and the DNA damages and modifications of their cell surface markers were assessed and statistically evaluated over several days. The cells survived the transfer procedure with a rate of 98%  +/- 1% standard error of the mean (skin cells) and 90%  +/- 10% (hMSC), respectively. All used cell types maintain their ability to proliferate after LIFT. Further, skin cells and hMSC did not show an increase of apoptosis or DNA fragmentation. In addition, the hMSC keep their phenotype as proven by fluorescence activated cell sorting (FACS) analysis. This study demonstrates LIFT as a suitable technique for unharmed computer-controlled positioning of different cell types and a promising tool for future applications in the ex vivo generation of tissue replacements.

  20. Human spleen and red blood cells

    Science.gov (United States)

    Pivkin, Igor; Peng, Zhangli; Karniadakis, George; Buffet, Pierre; Dao, Ming

    2016-11-01

    Spleen plays multiple roles in the human body. Among them is removal of old and altered red blood cells (RBCs), which is done by filtering cells through the endothelial slits, small micron-sized openings. There is currently no experimental technique available that allows us to observe RBC passage through the slits. It was previously noticed that people without a spleen have less deformable red blood cells, indicating that the spleen may play a role in defining the size and shape of red blood cells. We used detailed RBC model implemented within the Dissipative Particle Dynamics (DPD) simulation framework to study the filter function of the spleen. Our results demonstrate that spleen indeed plays major role in defining the size and shape of the healthy human red blood cells.

  1. Induction of anti-leukemic cytotoxic T lymphocytes by fusion of patient-derived dendritic cells with autologous myeloblasts.

    Science.gov (United States)

    Gong, Jianlin; Koido, Shigeo; Kato, Yoko; Tanaka, Yasuhiro; Chen, Dongshu; Jonas, Anna; Galinsky, Ilene; DeAngelo, Daniel; Avigan, David; Kufe, Donald; Stone, Richard

    2004-12-01

    Presentation of AML antigens by dendritic cells (DC) could potentially induce a T cell-mediated anti-leukemic immune response. In the present study, we generated DC from adherent (AD-DC) and non-adherent (NAD-DC) myeloblasts obtained from bone marrows of AML patients. Both cell populations displayed morphological, phenotypic and functional properties of DC. The functions of NAD-DC were compared to AD-DC that had been fused with autologous AML blasts (FC/AML). The FC/AML induced greater T cell proliferation and CTL activity against autologous AML blasts (9/10 cases) as compared to NAD-DC. FC/AML may thus represent a promising strategy for DC-based immunotherapy of patients with AML.

  2. Rotating cell culture systems for human cell culture: human trophoblast cells as a model.

    Science.gov (United States)

    Zwezdaryk, Kevin J; Warner, Jessica A; Machado, Heather L; Morris, Cindy A; Höner zu Bentrup, Kerstin

    2012-01-18

    The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines support our understanding of invasion of the uterine wall and remodeling of uterine spiral arteries by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS. The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies. The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS

  3. Autophagy in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Thien Tra

    Full Text Available Autophagy (macroautophagy is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of embryoid bodies. The precise roles of autophagy during early human embryonic development, remain however largely uncharacterized. Since human embryonic stem cells constitute a unique model system to study early human embryogenesis we investigated the occurrence of autophagy in human embryonic stem cells. We have, using lentiviral transduction, established multiple human embryonic stem cell lines that stably express GFP-LC3, a fluorescent marker for the autophagosome. Each cell line displays both a normal karyotype and pluripotency as indicated by the presence of cell types representative of the three germlayers in derived teratomas. GFP expression and labelling of autophagosomes is retained after differentiation. Baseline levels of autophagy detected in cultured undifferentiated hESC were increased or decreased in the presence of rapamycin and wortmannin, respectively. Interestingly, autophagy was upregulated in hESCs induced to undergo differentiation by treatment with type I TGF-beta receptor inhibitor SB431542 or removal of MEF secreted maintenance factors. In conclusion we have established hESCs capable of reporting macroautophagy and identify a novel link between autophagy and early differentiation events in hESC.

  4. Human embryonic stem cells for neuronal repair.

    Science.gov (United States)

    Ben-Hur, Tamir

    2006-02-01

    Human embryonic stem cells may serve as a potentially endeless source of transplantable cells to treat various neurologic disorders. Accumulating data have shown the therapeutic value of various neural precursor cell types in experimental models of neurologic diseases. Tailoring cell therapy for specific disorders requires the generation of cells that are committed to specific neural lineages. To this end, protocols were recently developed for the derivation of dopaminergic neurons, spinal motor neurons and oligodendrocytes from hESC. These protocols recapitulate normal development in culture conditions. However, a novel concept emerging from these studies is that the beneficial effect of transplanted stem cells is not only via cell replacement in damaged host tissue, but also by trophic and protective effects, as well as by an immunomodulatory effect that down-regulates detrimental brain inflammation.

  5. Mutation patterns of 16 genes in primary and secondary acute myeloid leukemia (AML with normal cytogenetics.

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    Marta Fernandez-Mercado

    Full Text Available Acute myeloid leukemia patients with normal cytogenetics (CN-AML account for almost half of AML cases. We aimed to study the frequency and relationship of a wide range of genes previously reported as mutated in AML (ASXL1, NPM1, FLT3, TET2, IDH1/2, RUNX1, DNMT3A, NRAS, JAK2, WT1, CBL, SF3B1, TP53, KRAS and MPL in a series of 84 CN-AML cases. The most frequently mutated genes in primary cases were NPM1 (60.8% and FLT3 (50.0%, and in secondary cases ASXL1 (48.5% and TET2 (30.3%. We showed that 85% of CN-AML patients have mutations in at least one of ASXL1, NPM1, FLT3, TET2, IDH1/2 and/or RUNX1. Serial samples from 19 MDS/CMML cases that progressed to AML were analyzed for ASXL1/TET2/IDH1/2 mutations; seventeen cases presented mutations of at least one of these genes. However, there was no consistent pattern in mutation acquisition during disease progression. This report concerns the analysis of the largest number of gene mutations in CN-AML studied to date, and provides insight into the mutational profile of CN-AML.

  6. Mutation patterns of 16 genes in primary and secondary acute myeloid leukemia (AML) with normal cytogenetics.

    Science.gov (United States)

    Fernandez-Mercado, Marta; Yip, Bon Ham; Pellagatti, Andrea; Davies, Carwyn; Larrayoz, María José; Kondo, Toshinori; Pérez, Cristina; Killick, Sally; McDonald, Emma-Jane; Odero, María Dolores; Agirre, Xabier; Prósper, Felipe; Calasanz, María José; Wainscoat, James S; Boultwood, Jacqueline

    2012-01-01

    Acute myeloid leukemia patients with normal cytogenetics (CN-AML) account for almost half of AML cases. We aimed to study the frequency and relationship of a wide range of genes previously reported as mutated in AML (ASXL1, NPM1, FLT3, TET2, IDH1/2, RUNX1, DNMT3A, NRAS, JAK2, WT1, CBL, SF3B1, TP53, KRAS and MPL) in a series of 84 CN-AML cases. The most frequently mutated genes in primary cases were NPM1 (60.8%) and FLT3 (50.0%), and in secondary cases ASXL1 (48.5%) and TET2 (30.3%). We showed that 85% of CN-AML patients have mutations in at least one of ASXL1, NPM1, FLT3, TET2, IDH1/2 and/or RUNX1. Serial samples from 19 MDS/CMML cases that progressed to AML were analyzed for ASXL1/TET2/IDH1/2 mutations; seventeen cases presented mutations of at least one of these genes. However, there was no consistent pattern in mutation acquisition during disease progression. This report concerns the analysis of the largest number of gene mutations in CN-AML studied to date, and provides insight into the mutational profile of CN-AML.

  7. Implication of Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Interleukin-3 (IL-3) in Children with Acute Myeloid Leukaemia (AML).

    Science.gov (United States)

    Elbaz, O; Shaltout, A

    2000-01-01

    Granulocyte-macrophage colony stimulating factor (GM-CSF) and Interleukin-3 (IL-3) are increasingly used to stimulate granulopoiesis in neutropenic patients but these are rarely used in the lights of knowledge of the endogenous CSF-levels. In this study we measured serum levels of GM-CSF and IL-3 at diagnosis and after remission in children with acute leukaemia, using an enzyme linked immuno-sorbent assay (ELISA) techniques in 14 patients with acute myeloid leukaemia (AML) and 27 patients with acute lymphoblastic leukaemia (ALL). Twelve healthy age-matched children were used as a reference group. AML patients showed a highly significant increase in serum levels of GM-CSF and IL-3 before induction of therapy (p 0.5), with no significant difference between preinduction and postinduction serum levels of either (p > 0.5). Since these cytokines are known to be fundamental for the growth of AML cells, we postulate that the pretreatment levels of both GM-CSF and IL-3 could play a role in the pathogenesis of AML.

  8. Implication of Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Interleukin-3 (IL-3) in Children with Acute Myeloid Leukaemia (AML); Malignancy.

    Science.gov (United States)

    Elbaz, Osama; Shaltout, Ali

    2001-01-01

    Granulocyte-macrophage colony stimulating factor (GM-CSF) and Interleukin-3 (IL-3) are increasingly used to stimulate granulopoiesis in neutropenic patients but these are rarely used in the lights of knowledge of the endogenous CSF-levels. In this study we measured serum levels of GM-CSF and IL-3 at diagnosis and after remission in children with acute leukaemia, using an enzyme linked immuno-sorbent assay (ELISA) techniques in 14 patients with acute myeloid leukaemia (AML) and 27 patients with acute lymphoblastic leukaemia (ALL). Twelve healthy age-matched children were used as a reference group. AML patients showed a highly significant increase in serum levels of GM-CSF and IL-3 before induction of therapy (p 0.5), with no significant difference between preinduction and postinduction serum levels of either (p > 0.5). Since these cytokines are known to be fundamental for the growth of AML cells, we postulate that the pretreatment levels of both GM-CSF and IL-3 could play a role in the pathogenesis of AML.

  9. Mitotic crossover promotes leukemogenesis in children born with TEL-AML1 via the generation of loss of heterozygosity at 12p

    Directory of Open Access Journals (Sweden)

    Ivan Ivanovski

    2015-11-01

    Full Text Available TEL-AML1 (ETV6-RUNX1 fusion gene which is formed prenatally in 1% of the newborns, is a common genetic abnormality in childhood Bcell precursor acute lymphoblastic leukemia. But only one child out of a hundred children born with this fusion gene develops leukemia (bottleneck phenomenon later in its life, if contracts the second mutation. In other words, out of a hundred children born with TEL-AML1 only one child is at risk for leukemia development, which means that TEL-AML1 fusion gene is not sufficient for overt leukemia. There is a stringent requirement for a second genetic abnormality for leukemia development and this is the real or the ultimate cause of the leukemia bottleneck phenomenon. In most cases of TEL-AML1+ leukemia, the translocation t(12;21 is complemented with the loss of the normal TEL gene, not involved in the translocation, on the contralateral 12p. The loss of the normal TEL gene, i.e. loss of heterozygosity at 12p, occurs postnatally during the mitotic proliferation of TEL-AML1+ cell in the mitotic crossing over process. Mitotic crossing over is a very rare event with a frequency rate of 10–6 in a 10 kb region. The exploration and identification of the environmental exposure(s that cause(s proliferation of the TELAML1+ cell in which approximately 106 mitoses are generated to cause 12p loss of heterozygosity, i.e. TEL gene deletion, may contribute to the introduction of preventive measures for leukemia.

  10. 3 CFR - Guidelines for Human Stem Cell Research

    Science.gov (United States)

    2010-01-01

    ... 3 The President 1 2010-01-01 2010-01-01 false Guidelines for Human Stem Cell Research Presidential Documents Other Presidential Documents Memorandum of July 30, 2009 Guidelines for Human Stem Cell Research..., scientifically worthy human stem cell research, including human embryonic stem cell research, to the extent...

  11. Myristoylation profiling in human cells and zebrafish

    Directory of Open Access Journals (Sweden)

    Malgorzata Broncel

    2015-09-01

    Full Text Available Human cells (HEK 293, HeLa, MCF-7 and zebrafish embryos were metabolically tagged with an alkynyl myristic acid probe, lysed with an SDS buffer and tagged proteomes ligated to multifunctional capture reagents via copper-catalyzed alkyne azide cycloaddition (CuAAC. This allowed for affinity enrichment and high-confidence identification, by delivering direct MS/MS evidence for the modification site, of 87 and 61 co-translationally myristoylated proteins in human cells and zebrafish, respectively. The data have been deposited to ProteomeXchange Consortium (Vizcaíno et al., 2014 Nat. Biotechnol., 32, 223–6 (PXD001863 and PXD001876 and are described in detail in Multifunctional reagents for quantitative proteome-wide analysis of protein modification in human cells and dynamic protein lipidation during vertebrate development׳ by Broncel et al., Angew. Chem. Int. Ed.

  12. Establishment of Human Neural Progenitor Cells from Human Induced Pluripotent Stem Cells with Diverse Tissue Origins

    Science.gov (United States)

    Fukusumi, Hayato; Shofuda, Tomoko; Bamba, Yohei; Yamamoto, Atsuyo; Kanematsu, Daisuke; Handa, Yukako; Okita, Keisuke; Nakamura, Masaya; Yamanaka, Shinya; Okano, Hideyuki; Kanemura, Yonehiro

    2016-01-01

    Human neural progenitor cells (hNPCs) have previously been generated from limited numbers of human induced pluripotent stem cell (hiPSC) clones. Here, 21 hiPSC clones derived from human dermal fibroblasts, cord blood cells, and peripheral blood mononuclear cells were differentiated using two neural induction methods, an embryoid body (EB) formation-based method and an EB formation method using dual SMAD inhibitors (dSMADi). Our results showed that expandable hNPCs could be generated from hiPSC clones with diverse somatic tissue origins. The established hNPCs exhibited a mid/hindbrain-type neural identity and uniform expression of neural progenitor genes. PMID:27212953

  13. Establishment of Human Neural Progenitor Cells from Human Induced Pluripotent Stem Cells with Diverse Tissue Origins.

    Science.gov (United States)

    Fukusumi, Hayato; Shofuda, Tomoko; Bamba, Yohei; Yamamoto, Atsuyo; Kanematsu, Daisuke; Handa, Yukako; Okita, Keisuke; Nakamura, Masaya; Yamanaka, Shinya; Okano, Hideyuki; Kanemura, Yonehiro

    2016-01-01

    Human neural progenitor cells (hNPCs) have previously been generated from limited numbers of human induced pluripotent stem cell (hiPSC) clones. Here, 21 hiPSC clones derived from human dermal fibroblasts, cord blood cells, and peripheral blood mononuclear cells were differentiated using two neural induction methods, an embryoid body (EB) formation-based method and an EB formation method using dual SMAD inhibitors (dSMADi). Our results showed that expandable hNPCs could be generated from hiPSC clones with diverse somatic tissue origins. The established hNPCs exhibited a mid/hindbrain-type neural identity and uniform expression of neural progenitor genes.

  14. Gene expression profiling in MDS and AML: potential and future avenues

    DEFF Research Database (Denmark)

    Theilgaard-Mönch, K; Boultwood, J; Ferrari, S

    2011-01-01

    Today, the classification systems for myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) already incorporate cytogenetic and molecular genetic aberrations in an attempt to better reflect disease biology. However, in many MDS/AML patients no genetic aberrations have been identified yet...... the MDS/AML classification system, incorporating as yet unknown molecular genetic and epigenetic pathomechanisms, which are likely reflected by aberrant gene expression patterns. In this study, we provide a comprehensive review on how GEP has contributed to a refined molecular taxonomy of MDS and AML...... technologies, which will continue to improve our understanding of malignant transformation in myeloid malignancies and thereby contribute to individualized risk-adapted treatment strategies for MDS and AML patients....

  15. Efficient derivation and genetic modifications of human pluripotent stem cells on engineered human feeder cell lines.

    Science.gov (United States)

    Zou, Chunlin; Chou, Bin-Kuan; Dowey, Sarah N; Tsang, Kitman; Huang, Xiaosong; Liu, Cyndi F; Smith, Cory; Yen, Jonathan; Mali, Prashant; Zhang, Yu Alex; Cheng, Linzhao; Ye, Zhaohui

    2012-08-10

    Derivation of pluripotent stem cells (iPSCs) induced from somatic cell types and the subsequent genetic modifications of disease-specific or patient-specific iPSCs are crucial steps in their applications for disease modeling as well as future cell and gene therapies. Conventional procedures of these processes require co-culture with primary mouse embryonic fibroblasts (MEFs) to support self-renewal and clonal growth of human iPSCs as well as embryonic stem cells (ESCs). However, the variability of MEF quality affects the efficiencies of all these steps. Furthermore, animal sourced feeders may hinder the clinical applications of human stem cells. In order to overcome these hurdles, we established immortalized human feeder cell lines by stably expressing human telomerase reverse transcriptase, Wnt3a, and drug resistance genes in adult mesenchymal stem cells. Here, we show that these immortalized human feeders support efficient derivation of virus-free, integration-free human iPSCs and long-term expansion of human iPSCs and ESCs. Moreover, the drug-resistance feature of these feeders also supports nonviral gene transfer and expression at a high efficiency, mediated by piggyBac DNA transposition. Importantly, these human feeders exhibit superior ability over MEFs in supporting homologous recombination-mediated gene targeting in human iPSCs, allowing us to efficiently target a transgene into the AAVS1 safe harbor locus in recently derived integration-free iPSCs. Our results have great implications in disease modeling and translational applications of human iPSCs, as these engineered human cell lines provide a more efficient tool for genetic modifications and a safer alternative for supporting self-renewal of human iPSCs and ESCs.

  16. Optimization of Human NK Cell Manufacturing: Fully Automated Separation, Improved Ex Vivo Expansion Using IL-21 with Autologous Feeder Cells, and Generation of Anti-CD123-CAR-Expressing Effector Cells.

    Science.gov (United States)

    Klöß, Stephan; Oberschmidt, Olaf; Morgan, Michael; Dahlke, Julia; Arseniev, Lubomir; Huppert, Volker; Granzin, Markus; Gardlowski, Tanja; Matthies, Nadine; Soltenborn, Stephanie; Schambach, Axel; Koehl, Ulrike

    2017-10-01

    The administration of ex vivo expanded natural killer (NK) cells as potential antitumor effector cells appears to be suitable for effector cell-based immunotherapies in high-risk cancer patients. However, good manufacturing practice (GMP)-compliant manufacturing of clinical-grade NK cells at sufficiently high numbers represents a great challenge. Therefore, previous expansion protocols for those effector cells were improved and optimized by using newly developed culture medium, interleukin (IL)-21, and autologous feeder cells (FCs). Separation of primary human NK cells (CD56(+)CD3(-)) was carried out with the CliniMACS Prodigy(®) in a single process, starting with approximately 1.2 × 10(9) leukocytes collected by small-scale lymphapheresis or from buffy coats. Enriched NK cells were adjusted to starting cell concentrations within approximately 1 × 10(6) effector cells/mL and cultured in comparative expansion experiments for 14 days with IL-2 (1,000 IU/mL) in different GMP-compliant media (X-VIVO(™)10, CellGro(®), TexMACS(™), and NK MACS(®)). After medium optimization, beneficial effects for functionality and phenotype were investigated at the beginning of cell expansion with irradiated (25 Gy) autologous FCs at a ratio of 20:1 (feeder: NK) in the presence or absence of IL-21 (100 ng/mL). Additionally, expanded NK cells were gene modified to express chimeric antigen receptors (CARs) against CD123, a common marker for acute myeloid leukemia (AML). Cytotoxicity, degranulation, and cytokine release of transduced NK cells were determined against KG1a cells in flow cytometric analysis and fluorescent imaging. The Prodigy manufacturing process revealed high target cell viabilities (median 95.4%), adequate NK cell recovery (median 60.4%), and purity of 95.4% in regard to CD56(+)CD3(-) target cells. The process in its early phase of development led to a median T-cell depletion of log 3.5 after CD3 depletion and log 3.6 after the whole process, including

  17. [Human pluripotent stem cell and neural differentiation].

    Science.gov (United States)

    Wataya, Takafumi; Muguruma, Keiko; Sasai, Yoshiki

    2008-10-01

    Recovery of lost brain function is an important issue in medical studies because neurons of the central nervous system (CNS) have poor potential for regeneration. Since few CNS diseases can be treated completely by medicines, regenerative therapy by using stem cells should be studied as a new type of therapeutic intervention. The efficacy of cell replacement therapy in Parkinson's disease has been well investigated. Several studies on fetal tissue transplantation have revealed that quantity and purity of transplanted cells are necessary for recovery of symptoms. SFEB (Serum-free floating culture of embryoid body-like aggregates) method is capable of inducing multi-potential CNS progenitors that can be steered to differentiate into region-specific tissues. On the basis of the existing knowledge of embryology, we have succeeded in the generating of various types of neurons such as telencephalic, cerebeller (Purkinje and granule cells), retinal (photoreceptor cells) and hypothalamic neurons. Application of this culture method to human ES (hES) cells is necessary for clinical purpose: however, poor survival of hES cells in SFEB culture might limit the possibility of using these cells for future medical applications. We found that a selective Rho-associated kinase (ROCK) inhibitor, Y-27632, markedly diminished the dissociation-induced apoptosis of hES cells and enabled the cells to form aggregates in SFEB culture. For both mouse and human ES cells, SFEB culture is a favorable method that can generate large amounts of region-specific neurons. However, stem cell-based therapy continues to face several obstacles. It is important that researchers in the basic sciences and clinical medicine should discuss these problems together to overcome both scientific and ethical issues related to stem cells.

  18. The Broad Anti-AML Activity of the CD33/CD3 BiTE Antibody Construct, AMG 330, Is Impacted by Disease Stage and Risk.

    Directory of Open Access Journals (Sweden)

    Kimberly H Harrington

    Full Text Available The CD33/CD3-bispecific T-cell engaging (BiTE antibody construct, AMG 330, potently lyses CD33+ leukemic cells in vitro. Using specimens from 41 patients with acute myeloid leukemia (AML, we studied the factors that might contribute to clinical response or resistance. For this purpose, thawed aliquots of primary AML samples were immunophenotypically characterized and subjected to various doses of AMG 330 in the presence or absence of healthy donor T-cells. After 48 hours, drug-specific cytotoxicity was quantified and correlated with CD33 expression levels, amounts of T-cells present, and other disease characteristics. AMG 330 caused modest cytotoxicity that was correlated with the amount of autologous T-cells (P = 0.0001 but not CD33 expression, as AMG 330 exerted marked cytotoxic effects in several specimens with minimal CD33 expression. With healthy donor T-cells added, AMG 330 cytotoxicity depended on the drug dose and effector:target (E:T cell ratio. High cytotoxic activity was observed even with minimal CD33 expression, and AMG 330 cytotoxicity and CD33 expression correlated only at high E:T cell ratio and high AMG 330 doses (P<0.003. AMG 330 resulted in significantly higher cytotoxicity in specimens from patients with newly diagnosed AML than those with relapsed/refractory disease despite similar levels of CD33 on myeloblasts. AMG 330 cytotoxicity also appeared greater in specimens from patients with favorable-risk disease as compared to other specimens. Together, our data demonstrate that AMG 330 is highly active in primary AML specimens across the entire disease spectrum, while suggesting the presence of yet undefined, CD33-independent, relative resistance mechanisms in specific patient subsets.

  19. T-cell response in human leishmaniasis

    DEFF Research Database (Denmark)

    Kharazmi, A; Kemp, K; Ismail, A

    1999-01-01

    In the present communication we provide evidence for the existence of a Th1/Th2 dichotomy in the T-cell response to Leishmania antigens in human leishmaniasis. Our data suggest that the pattern of IL-4 and IFN-gamma response is polarised in these patients. Lymphocytes from individuals recovered......+. Furthermore, IL-10 plays an important role in the development of post kala azar dermal leishmaniasis (PKDL) from VL. The balance between the parasitic-specific T-cell response plays an important regulatory role in determining the outcome of Leishmania infections in humans....

  20. Characterization of human pluripotent stem cells.

    Science.gov (United States)

    Gokhale, Paul J; Andrews, Peter W

    2013-12-18

    Human pluripotent stem cells (PSCs), whether embryonic stem cells or induced PSCs, offer enormous opportunities for regenerative medicine and other biomedical applications once we have developed the ability to harness their capacity for extensive differentiation. Central to this is our ability to identify and characterize such PSCs, but this is fraught with potential difficulties that arise from a tension between functional definitions of pluripotency and the more convenient use of 'markers', a problem exacerbated by ethical issues, our lack of knowledge of early human embryonic development, and differences from the mouse paradigm.

  1. CLOSTRIDIUM SPORE ATTACHMENT TO HUMAN CELLS

    Energy Technology Data Exchange (ETDEWEB)

    PANESSA-WARREN,B.; TORTORA,G.; WARREN,J.

    1997-08-10

    This paper uses high resolution scanning electron microscopy (SEM) with a LaB6 gun and the newest commercial field emission guns, to obtain high magnification images of intact clostridial spores throughout the activation/germination/outgrowth process. By high resolution SEM, the clostridial exosporial membrane can be seen to produce numerous delicate projections (following activation), that extend from the exosporial surface to a nutritive substrate (agar), or cell surface when anaerobically incubated in the presence of human cells (embryonic fibroblasts and colon carcinoma cells). Magnifications of 20,000 to 200,000Xs at accelerating voltages low enough to minimize or eliminate specimen damage (1--5 kV) have permitted the entire surface of C.sporogenes and C.difficile endospores to be examined during all stages of germination. The relationships between the spore and the agar or human cell surface were also clearly visible.

  2. Human pluripotent stem cells in contemporary medicine

    Directory of Open Access Journals (Sweden)

    S. A. Rodin

    2015-01-01

    Full Text Available Human pluripotent stem cells (hPSCs are capable of indefinite proliferation and can be differentiated into any cell type of the human body. Therefore, they are a promising source of cells for treatment of numerous degenerative diseases and injuries. Pluripotent stem cells are also associated with a number of ethical, safety and technological issues. In this review, we describe various types of hPSCs, safety issues that concern all or some types of hPSCs and methods of clinical-grade hPSC line development. Also, we discuss current and past clinical trials involving hPSCs, their outcomes and future perspectives of hPSC-based therapy. 

  3. Cell pattern in adult human corneal endothelium.

    Directory of Open Access Journals (Sweden)

    Carlos H Wörner

    Full Text Available A review of the current data on the cell density of normal adult human endothelial cells was carried out in order to establish some common parameters appearing in the different considered populations. From the analysis of cell growth patterns, it is inferred that the cell aging rate is similar for each of the different considered populations. Also, the morphology, the cell distribution and the tendency to hexagonallity are studied. The results are consistent with the hypothesis that this phenomenon is analogous with cell behavior in other structures such as dry foams and grains in polycrystalline materials. Therefore, its driving force may be controlled by the surface tension and the mobility of the boundaries.

  4. Merkel cell distribution in the human eyelid

    Directory of Open Access Journals (Sweden)

    C.A. May

    2013-10-01

    Full Text Available Although Merkel cell carcinoma of the eye lid is reported frequently in the literature, only limited information exists about the distribution of Merkel cells in this tissue. Therefore, serial sections of 18 human cadaver eye lids (donors ages ranging between 63 and 97 years were stained for cytokeratin 20 in various planes. The overall appearance of Merkel cells in these samples was low and mainly located in the outer root layer of the cilia hair follicles. Merkel cells were more frequent in the middle, and almost not detectable at the nasal and temporal edges. The localization is in accordance with that of Merkel cell carcinoma, but concerning the scarce appearance within this adulthood group, a specific physiological role of these cells in the eye lid is difficult to establish.

  5. Natural killer cells in human autoimmune disorders

    Science.gov (United States)

    2013-01-01

    Natural killer (NK) cells are innate lymphocytes that play a critical role in early host defense against viruses. Through their cytolytic capacity and generation of cytokines and chemokines, NK cells modulate the activity of other components of the innate and adaptive immune systems and have been implicated in the initiation or maintenance of autoimmune responses. This review focuses on recent research elucidating a potential immunoregulatory role for NK cells in T-cell and B-cell-mediated autoimmune disorders in humans, with a particular focus on multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematous. A better understanding of the contributions of NK cells to the development of autoimmunity may lead to novel therapeutic targets in these diseases. PMID:23856014

  6. Human Colon Cancer Cells Cultivated in Space

    Science.gov (United States)

    1995-01-01

    Within five days, bioreactor cultivated human colon cancer cells (shown) grown in Microgravity on the STS-70 mission in 1995, had grown 30 times the volume of the control specimens on Earth. The samples grown in space had a higher level of cellular organization and specialization. Because they more closely resemble tumors found in the body, microgravity grown cell cultures are ideal for research purposes.

  7. WT1 vaccination in AML and MDS: A pilot trial with synthetic analog peptides.

    Science.gov (United States)

    Brayer, Jason; Lancet, Jeffrey E; Powers, John; List, Alan; Balducci, Lodovico; Komrokji, Rami; Pinilla-Ibarz, Javier

    2015-07-01

    Peptide vaccines are capable of eliciting immune responses targeting tumor-associated antigens such as the Wilms' Tumor 1 (WT1) antigen, often overexpressed in myeloid malignancies. Here, we assessed the safety, tolerability, and immunogenicity of a polyvalent WT1 peptide vaccine. Individuals with WT1-positive acute myeloid leukemia (AML) in first (CR1) or second (CR2) remission or with higher-risk myelodysplastic syndrome (MDS) following at least 1 prior line of therapy were vaccinated with a mixture of peptides derived from the WT1 protein, with sargramostim injections before vaccination to amplify immunogenicity. Six vaccinations were delivered biweekly, continuing then monthly until patients received 12 vaccinations or showed disease relapse or progression. Therapeutic efficacy was evaluated by progression-free and overall survival. Immune responses were evaluated by delayed-type hypersensitivity testing and T-cell IFNγ ELISPOT at specified intervals. In 16 patients who received at least one vaccination, 10 completed the planned course of six vaccinations and six continued for up to six additional monthly vaccinations. Vaccinations were well tolerated, with no patients discontinuing due to toxicity. One of two patients with high-risk MDS experienced a prolonged decrease in transfusion dependence. Two of 14 AML patients demonstrated relapse-free survival >1 year. Both patients were in CR2 at time of vaccination, with duration of their remission exceeding duration of their first remission, suggesting a potential benefit. Our WT1 vaccine was well-tolerated. The clinical benefit that we observed in several patients suggests engagement of a protective immune response, indicating a need for further trials.

  8. Quercetin Inhibits Cell Migration and Invasion in Human Osteosarcoma Cells

    Directory of Open Access Journals (Sweden)

    Haifeng Lan

    2017-09-01

    Full Text Available Background/Aims: Osteosarcoma is a malignant tumor associated with high mortality; however, no effective therapies for the disease have been developed. Several studies have focused on elucidating the pathogenesis of osteosarcoma and have aimed to develop novel therapies for the disease. Quercetin is a vital dietary flavonoid that has been shown to have a variety of anticancer effects, as it induces cell cycle arrest, apoptosis, and differentiation and is involved in cell adhesion, metastasis and angiogenesis. Herein, we aimed to investigate the effects of quercetin on osteosarcoma migration and invasion in vitro and in vivo and to explore the molecular mechanisms underlying its effects on osteosarcoma migration and invasion. Methods: Cell viability, cell cycle activity and cell apoptosis were measured using CCK-8 assay and flow cytometry, and cell migration and invasion were evaluated by wound healing and transwell assays, respectively. The mRNA and protein expression levels of several proteins of interest were assessed by real-time quantitative PCR and western blotting, respectively. Moreover, a nude mouse model of human osteosarcoma lung metastasis was established to assess the anti-metastatic effects of quercetin in vivo. Results: We noted no significant differences in cell cycle activity and apoptosis between HOS and MG63 cells and control cells. Treatment with quercetin significantly attenuated cell migration and invasion in HOS and MG63 cells compared with treatment with control medium. Moreover HIF-1α, VEGF, MMP2, and MMP9 mRNA and protein expression levels were significantly downregulated in HOS cells treated with quercetin compared with HOS cells treated with controls. Additionally, treatment with quercetin attenuated metastatic lung tumor formation and growth in the nude mouse model of osteosarcoma compared with treatment with controls. Conclusion: Our findings regarding the inhibitory effects of quercetin on cell migration and

  9. Human Mammary Luminal Epithelial Cells Contain Progenitors to Myoepithelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Pechoux, Christine; Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Bissell, Mina J; Petersen, Ole

    1999-02-01

    The origin of the epithelial and myoepithelial cells in the human breast has not been delineated. In this study we have addressed whether luminal epithelial cells and myoepithelial cells are vertically connected, i.e., whether one is the precursor for the other. We used a primary culture assay allowing preservation of basic phenotypic traits of luminal epithelial and myoepithelial cells in culture. The two cell types were then separated immunomagnetically using antibodies directed against lineage-specific cell surface antigens into at best 100% purity. The cellular identity was ascertained by cytochemistry, immunoblotting, and 2-D gel electrophoresis. Luminal epithelial cells were identified by strong expression of cytokeratins 18 and 19 while myoepithelial cells were recognized by expression of vimentin and {alpha}-smooth muscle actin. We used a previously devised culture medium (CDM4) that allows vigorous expansion of proliferative myoepithelial cells and also devised a medium (CDM6) that allowed sufficient expansion of differentiated luminal epithelial cells based on addition of hepatocyte growth factor/scatter factor. The two different culture media supported each lineage for at least five passages without signs of interconversion. We used parallel cultures where we switched culture media, thus testing the ability of each lineage to convert to the other. Whereas the myoepithelial lineage showed no signs of interconversion, a subset of luminal epithelial cells, gradually, but distinctly, converted to myoepithelial cells. We propose that in the mature human breast, it is the luminal epithelial cell compartment that gives rise to myoepithelial cells rather than the other way around.

  10. Genetic Manipulation of Human Embryonic Stem Cells.

    Science.gov (United States)

    Eiges, Rachel

    2016-01-01

    One of the great advantages of embryonic stem (ES) cells over other cell types is their accessibility to genetic manipulation. They can easily undergo genetic modifications while remaining pluripotent, and can be selectively propagated, allowing the clonal expansion of genetically altered cells in culture. Since the first isolation of ES cells in mice, many effective techniques have been developed for gene delivery and manipulation of ES cells. These include transfection, electroporation, and infection protocols, as well as different approaches for inserting, deleting, or changing the expression of genes. These methods proved to be extremely useful in mouse ES cells, for monitoring and directing differentiation, discovering unknown genes, and studying their function, and are now being extensively implemented in human ES cells (HESCs). This chapter describes the different approaches and methodologies that have been applied for the genetic manipulation of HESCs and their applications. Detailed protocols for generating clones of genetically modified HESCs by transfection, electroporation, and infection will be described, with special emphasis on the important technical details that are required for this purpose. All protocols are equally effective in human-induced pluripotent stem (iPS) cells.

  11. American experience in the implementation of AML / CFT system

    Directory of Open Access Journals (Sweden)

    Larin Dmitry

    2016-01-01

    Full Text Available This article presents a research into very specific matters and issues of creating and further improvement of Anti-Money Laundering and Combating the Financing of Terrorism system (abbreviated as AML/CFT in the USA. In contains a precise analysis of key elements of this system, as well as a deep look into the nature and purposes of financial intelligence “FinCEN”. Strong emphasis is laid on The US Foreign Account Tax Compliance Act (FATCA for the reason that, according to this Act, any foreign financial institution is legitimately forced to sign a special agreement with The US Internal Revenue Service (IRS, giving the Service power to maintain control over any American asset within its equity. The latter is understood as a legal right to investigate not only the accounts and property of American taxpayers, but to investigate any account or capital of any foreign institution having American shares or stocks.

  12. Modeling of C/EBPalpha mutant acute myeloid leukemia reveals a common expression signature of committed myeloid leukemia-initiating cells

    DEFF Research Database (Denmark)

    Kirstetter, Peggy; Schuster, Mikkel B; Bereshchenko, Oksana

    2008-01-01

    Mutations in the CEBPA gene are present in 7%-10% of human patients with acute myeloid leukemia (AML). However, no genetic models exist that demonstrate their etiological relevance. To mimic the most common mutations affecting CEBPA-that is, those leading to loss of the 42 kDa C/EBPalpha isoform (p...... penetrance. p42-deficient leukemia could be transferred by a Mac1+c-Kit+ population that gave rise only to myeloid cells in recipient mice. Expression profiling of this population against normal Mac1+c-Kit+ progenitors revealed a signature shared with MLL-AF9-transformed AML....

  13. High prognostic value of minimal residual disease detected by flow-cytometry-enhanced fluorescence in situ hybridization in core-binding factor acute myeloid leukemia (CBF-AML).

    Science.gov (United States)

    Wang, Libing; Gao, Lei; Xu, Sheng; Gong, Shenglan; Liu, Min; Qiu, Huiying; Xu, Xiaoqian; Ni, Xiong; Chen, Li; Lu, Shuqing; Chen, Jie; Song, Xianmin; Zhang, Weiping; Yang, Jianmin; Hu, Xiaoxia; Wang, Jianmin

    2014-10-01

    Acute myeloid leukemia (AML) is generally regarded as a disorder of stem cells, known as leukemic initiating cells (LICs), which initiate the disease and contribute to relapses. Although the phenotype of these cells remains unclear in most patients, they are enriched within the CD34(+)CD38(-) population. In core-binding factor (CBF) AML, the cytogenetic abnormalities also exist in LIC. The aim of this study was to determine the prognostic power of minimal residual disease (MRD) measured by fluorescence in situ hybridization (FISH) in CD34(+)CD38(-) cells sorted by flow cytometry at different periods during therapy. Thirty-six patients under 65 years of age with de novo CBF-AML treated with intensive chemotherapy were retrospectively included in this study. Correlations with relapse-free survival (RFS) and overall survival were evaluated with univariate and multivariate analyses. FISH efficiently identified LICs in the CD34(+)CD38(-) population. The presence of FISH(+)CD34(+)CD38(-) cells before consolidation was negatively associated with cumulative incidence of relapse (64 vs 18 %, P = .012), which showed prognostic value for RFS (12 vs 68 %, P = .008) and OS (11 vs 75 %, P = .0005), and retained prognostic significance for RFS in multivariate analysis. The detection of FISH(+)CD34(+)CD38(-) cells before consolidation therapy significantly correlated with long-term survival. Fluorescence-activated cell sorting (FACS)-FISH could be potentially adopted as a MRD monitor approach in clinical practice to identify CBF-AML patients at risk of treatment failure during therapy.

  14. Enriched retinal ganglion cells derived from human embryonic stem cells

    Science.gov (United States)

    Gill, Katherine P.; Hung, Sandy S. C.; Sharov, Alexei; Lo, Camden Y.; Needham, Karina; Lidgerwood, Grace E.; Jackson, Stacey; Crombie, Duncan E.; Nayagam, Bryony A.; Cook, Anthony L.; Hewitt, Alex W.; Pébay, Alice; Wong, Raymond C. B.

    2016-01-01

    Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment. Development of cell therapy requires a better understanding of the signals that direct stem cells into RGCs. Human embryonic stem cells (hESCs) represent an unlimited cellular source for generation of human RGCs in vitro. In this study, we present a 45-day protocol that utilises magnetic activated cell sorting to generate enriched population of RGCs via stepwise retinal differentiation using hESCs. We performed an extensive characterization of these stem cell-derived RGCs by examining the gene and protein expressions of a panel of neural/RGC markers. Furthermore, whole transcriptome analysis demonstrated similarity of the hESC-derived RGCs to human adult RGCs. The enriched hESC-RGCs possess long axons, functional electrophysiological profiles and axonal transport of mitochondria, suggestive of maturity. In summary, this RGC differentiation protocol can generate an enriched population of functional RGCs from hESCs, allowing future studies on disease modeling of optic neuropathies and development of cell therapies. PMID:27506453

  15. Human ES cells: starting culture from frozen cells.

    Science.gov (United States)

    Trish, Erin; Dimos, John; Eggan, Kevin

    2006-11-09

    Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock. First, a one to two day old ten cm plate of approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator. A frozen vial of cells from long term liquid nitrogen storage or a -80 C freezer is sourced and quickly submerged in a 37 C water bath for quick thawing. Cells in freezing media are then removed from the vial and placed in a large volume of HuES media. The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate. Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate. It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate. The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

  16. Sodium Valproate Induces Cell Senescence in Human Hepatocarcinoma Cells

    Directory of Open Access Journals (Sweden)

    Hong-Mei An

    2013-12-01

    Full Text Available Hepatocarcinogenesis is associated with epigenetic changes, including histone deacetylases (HDACs. Epigenetic modulation by HDAC inhibition is a potentially valuable approach for hepatocellular carcinoma treatment. In present study, we evaluated the anticancer effects of sodium valproate (SVP, a known HDAC inhibitor, in human hepatocarcinoma cells. The results showed SVP inhibited the proliferation of Bel-7402 cells in a dose-dependent manner. Low dose SVP treatment caused a large and flat morphology change, positive SA-β-gal staining, and G0/G1 phase cell cycle arrest in human hepatocarcinoma cells. Low dose SVP treatment also increased acetylation of histone H3 and H4 on p21 promoter, accompanied by up-regulation of p21 and down-regulation of RB phosphorylation. These observations suggested that a low dose of SVP could induce cell senescence in hepatocarcinoma cells, which might correlate with hyperacetylation of histone H3 and H4, up-regulation of p21, and inhibition of RB phosphorylation. Since the effective concentration inducing cell senescence in hepatocarcinoma cells is clinically available, whether a clinical dose of SVP could induce cell senescence in clinical hepatocarcinoma is worthy of further study.

  17. Human embryonic stem cell lines model experimental human cytomegalovirus latency.

    Science.gov (United States)

    Penkert, Rhiannon R; Kalejta, Robert F

    2013-05-28

    Herpesviruses are highly successful pathogens that persist for the lifetime of their hosts primarily because of their ability to establish and maintain latent infections from which the virus is capable of productively reactivating. Human cytomegalovirus (HCMV), a betaherpesvirus, establishes latency in CD34(+) hematopoietic progenitor cells during natural infections in the body. Experimental infection of CD34(+) cells ex vivo has demonstrated that expression of the viral gene products that drive productive infection is silenced by an intrinsic immune defense mediated by Daxx and histone deacetylases through heterochromatinization of the viral genome during the establishment of latency. Additional mechanistic details about the establishment, let alone maintenance and reactivation, of HCMV latency remain scarce. This is partly due to the technical challenges of CD34(+) cell culture, most notably, the difficulty in preventing spontaneous differentiation that drives reactivation and renders them permissive for productive infection. Here we demonstrate that HCMV can establish, maintain, and reactivate in vitro from experimental latency in cultures of human embryonic stem cells (ESCs), for which spurious differentiation can be prevented or controlled. Furthermore, we show that known molecular aspects of HCMV latency are faithfully recapitulated in these cells. In total, we present ESCs as a novel, tractable model for studies of HCMV latency.

  18. Favorable prognostic impact of NPM1 gene mutations in childhood acute myeloid leukemia, with emphasis on cytogenetically normal AML.

    NARCIS (Netherlands)

    Hollink, I.H.; Zwaan, C.M.; Zimmermann, M.; Arentsen-Peters, T.C.; Pieters, R.; Cloos, J.; Kaspers, G.J.L.; Graaf, S.S.N. de; Harbott, J.; Creutzig, U.; Reinhardt, D.; Heuvel-Eibrink, M.M. van den; Thiede, C.

    2009-01-01

    Nucleophosmin (NPM1) mutations occur frequently in adult cytogenetically normal acute myeloid leukemia (CN-AML) and confer favorable outcome. We investigated the frequency and prognostic significance of NPM1 mutations in childhood AML (n=298), specifically focusing on the CN-AML subgroup (n=100). Mu

  19. Phospho-MEK1/2 and uPAR Expression Determine Sensitivity of AML Blasts to a Urokinase-Activated Anthrax Lethal Toxin (PrAgU2/LF

    Directory of Open Access Journals (Sweden)

    Amira Bekdash

    2015-10-01

    Full Text Available In this study, we attempt to target both the urokinase plasminogen activator and the mitogen-activated protein kinase pathway in acute myeloid leukemia (AML cell lines and primary AML blasts using PrAgU2/LF, a urokinase-activated anthrax lethal toxin. PrAgU2/LF was cytotoxic to five out of nine AML cell lines. Cytotoxicity of PrAgU2/LF appeared to be nonapoptotic and was associated with MAPK activation and urokinase activity because all the PrAgU2/LF-sensitive cell lines showed both uPAR expression and high levels of MEK1/2 phosphorylation. Inhibition of uPAR or desensitization of cells to MEK1/2 inhibition blocked toxicity of PrAgU2/LF, indicating requirement for both uPAR expression and MAPK activation for activity. PrAgU2/LF was also cytotoxic to primary blasts from AML patients, with blasts from four out of five patients showing a cytotoxic response to PrAgU2/LF. Cytotoxicity of primary AML blasts was also dependent on uPAR expression and phos-MEK1/2 levels. CD34+ bone marrow blasts and peripheral blood mononuclear cells lacked uPAR expression and were resistant to PrAgU2/LF, demonstrating the lack of toxicity to normal hematological cells and, therefore, the tumor selectivity of this approach. Dose escalation in mice revealed that the maximal tolerated dose of PrAgU2/LF is at least 5.7-fold higher than that of the wild-type anthrax lethal toxin, PrAg/LF, further demonstrating the increased safety of this molecule. We have shown, in this study, that PrAgU2/LF is a novel, dual-specific molecule for the selective targeting of AML.

  20. A Natural Combination Extract of Viscum album L. Containing Both Triterpene Acids and Lectins Is Highly Effective against AML In Vivo.

    Directory of Open Access Journals (Sweden)

    Catharina I Delebinski

    Full Text Available Aqueous Viscum album L. extracts are widely used in complementary cancer medicine. Hydrophobic triterpene acids also possess anti-cancer properties, but due to their low solubility they do not occur in significant amounts in aqueous extracts. Using cyclodextrins we solubilised mistletoe triterpenes (mainly oleanolic acid and investigated the effect of a mistletoe whole plant extract on human acute myeloid leukaemia cells in vitro, ex vivo and in vivo. Single Viscum album L. extracts containing only solubilised triterpene acids (TT or lectins (viscum inhibited cell proliferation and induced apoptosis in a dose-dependent manner in vitro and ex vivo. The combination of viscum and TT extracts (viscumTT enhanced the induction of apoptosis synergistically. The experiments demonstrated that all three extracts are able to induce apoptosis via caspase-8 and -9 dependent pathways with down-regulation of members of the inhibitor of apoptosis and Bcl-2 families of proteins. Finally, the acute myeloid leukaemia mouse model experiment confirmed the therapeutic effectiveness of viscumTT-treatment resulting in significant tumour weight reduction, comparable to the effect in cytarabine-treated mice. These results suggest that the combination viscumTT may have a potential therapeutic value for the treatment AML.

  1. [Immune system evolution. (From cells to humans)].

    Science.gov (United States)

    Belek, A S

    1992-01-01

    The great variety of cells and molecules observed in the mammalian immune system can be explained by stepwise acquisition of them during phylogeny. Self/nonself discrimination and cell-mediated immunity have been present since the early stages of evolution. Although some inducible antimicrobial molecules have been demonstrated in invertebrates, immunoglobulins appear in vertebrates. T and B cell diversity, development of the lymphoid organs, MHC molecules, complement and cytokines are the characteristics that appear through the evolution of vertebrates. Further knowledge that will be obtained from phylogenetic studies will improve our understanding of the immune system of human.

  2. Centre for human development, stem cells & regeneration.

    Science.gov (United States)

    Oreffo, Richard O C

    2014-01-01

    The Centre for Human Development, Stem Cells and Regeneration (CHDSCR) was founded in 2004 as a cross-disciplinary research and translational program within the Faculty of Medicine at the University of Southampton. The Centre undertakes fundamental research into early development and stem cells together with applied translational research for patient benefit. The Centre has vibrant and thriving multidisciplinary research programs that harness the translational strength of the Faculty together with an innovative Stem Cell PhD program, outstanding clinical infrastructure and enterprise to deliver on this vision.

  3. Biobanking human embryonic stem cell lines

    DEFF Research Database (Denmark)

    Holm, Søren

    2016-01-01

    Stem cell banks curating and distributing human embryonic stem cells have been established in a number of countries and by a number of private institutions. This paper identifies and critically discusses a number of arguments that are used to justify the importance of such banks in policy...... are curiously absent from the particular stem cell banking policy discourse. This to some extent artificially isolates this discourse from the broader discussions about the flows of reproductive materials and tissues in modern society, and such isolation may lead to the interests of important actors being...

  4. Low-dose salinomycin induces anti-leukemic responses in AML and MLL.

    Science.gov (United States)

    Roulston, Gary D R; Burt, Charlotte L; Kettyle, Laura M J; Matchett, Kyle B; Keenan, Heather L; Mulgrew, Nuala M; Ramsey, Joanne M; Dougan, Caoifa; McKiernan, John; Grishagin, Ivan V; Mills, Ken I; Thompson, Alexander

    2016-11-08

    Development of anti-cancer drugs towards clinical application is costly and inefficient. Large screens of drugs, efficacious for non-cancer disease, are currently being used to identify candidates for repurposing based on their anti-cancer properties. Here, we show that low-dose salinomycin, a coccidiostat ionophore previously identified in a breast cancer screen, has anti-leukemic efficacy. AML and MLLr cell lines, primary cells and patient samples were sensitive to submicromolar salinomycin. Most strikingly, colony formation of normal hematopoietic cells was unaffected by salinomycin, demonstrating a lack of hemotoxicity at the effective concentrations. Furthermore, salinomycin treatment of primary cells resulted in loss of leukemia repopulation ability following transplantation, as demonstrated by extended recipient survival compared to controls. Bioinformatic analysis of a 17-gene signature identified and validated in primary MLLr cells, uncovered immunomodulatory pathways, hubs and protein interactions as potential transducers of low dose salinomycin treatment. Additionally, increased protein expression of p62/Sqstm1, encoded for by one of the 17 signature genes, demonstrates a role for salinomycin in aggresome/vesicle formation indicative of an autophagic response.Together, the data support the efficacy of salinomycin as an anti-leukemic at non-hemotoxic concentrations. Further investigation alone or in combination with other therapies is warranted for future clinical trial.

  5. ASXL2 mutations are frequently found in pediatric AML patients with t(8;21)/ RUNX1-RUNX1T1 and associated with a better prognosis.

    Science.gov (United States)

    Yamato, Genki; Shiba, Norio; Yoshida, Kenichi; Shiraishi, Yuichi; Hara, Yusuke; Ohki, Kentaro; Okubo, Jun; Okuno, Haruna; Chiba, Kenichi; Tanaka, Hiroko; Kinoshita, Akitoshi; Moritake, Hiroshi; Kiyokawa, Nobutaka; Tomizawa, Daisuke; Park, Myoung-Ja; Sotomatsu, Manabu; Taga, Takashi; Adachi, Souichi; Tawa, Akio; Horibe, Keizo; Arakawa, Hirokazu; Miyano, Satoru; Ogawa, Seishi; Hayashi, Yasuhide

    2017-05-01

    ASXL2 is an epigenetic regulator involved in polycomb repressive complex regulation or recruitment. Clinical features of pediatric acute myeloid leukemia (AML) patients with ASXL2 mutations remain unclear. Thus, we investigated frequencies of ASXL1 and ASXL2 mutations, clinical features of patients with these mutations, correlations of these mutations with other genetic alterations including BCOR/BCORL1 and cohesin complex component genes, and prognostic impact of these mutations in 369 pediatric patients with de novo AML (0-17 years). We identified 9 (2.4%) ASXL1 and 17 (4.6%) ASXL2 mutations in 25 patients. These mutations were more common in patients with t(8;21)(q22;q22)/RUNX1-RUNX1T1 (ASXL1, 6/9, 67%, P = 0.02; ASXL2, 10/17, 59%, P = 0.01). Among these 25 patients, 4 (27%) of 15 patients with t(8;21) and 6 (60%) of 10 patients without t(8;21) relapsed. However, most patients with relapse were rescued using stem cell transplantation irrespective of t(8;21). The overall survival (OS) and event-free survival (EFS) rates showed no differences among pediatric AML patients with t(8;21) and ASXL1 or ASXL2 mutations and ASXL wild-type (5-year OS, 75% vs. 100% vs. 91% and 5-year EFS, 67% vs. 80% vs. 67%). In 106 patients with t(8;21) AML, the coexistence of mutations in tyrosine kinase pathways and chromatin modifiers and/or cohesin complex component genes had no effect on prognosis. These results suggest that ASXL1 and ASXL2 mutations play key roles as cooperating mutations that induce leukemogenesis, particularly in pediatric AML patients with t(8;21), and these mutations might be associated with a better prognosis than that reported previously. © 2017 Wiley Periodicals, Inc.

  6. Advances in human B cell phenotypic profiling

    Directory of Open Access Journals (Sweden)

    Denise A Kaminski

    2012-10-01

    Full Text Available To advance our understanding and treatment of disease, research immunologists have been called-upon to place more centralized emphasis on impactful human studies. Such endeavors will inevitably require large-scale study execution and data management regulation (Big Biology, necessitating standardized and reliable metrics of immune status and function. A well-known example setting this large-scale effort in-motion is identifying correlations between eventual disease outcome and T lymphocyte phenotype in large HIV-patient cohorts using multiparameter flow cytometry. However, infection, immunodeficiency, and autoimmunity are also characterized by correlative and functional contributions of B lymphocytes, which to-date have received much less attention in the human Big Biology enterprise. Here, we review progress in human B cell phenotyping, analysis, and bioinformatics tools that constitute valuable resources for the B cell research community to effectively join in this effort.

  7. Allorestricted cytotoxic T cells specific for human CD45 show potent antileukemic activity.

    Science.gov (United States)

    Amrolia, Persis J; Reid, Steven D; Gao, Liquan; Schultheis, Beate; Dotti, Gianpietro; Brenner, Malcolm K; Melo, Junia V; Goldman, John M; Stauss, Hans J

    2003-02-01

    Recent advances have made haploidentical transplantation for leukemia feasible, but the rigorous T-cell depletion used contributes to the high relapse rates observed. We have attempted to improve the graft-versus-leukemia (GVL) effect by generating allorestricted cytotoxic T lymphocytes (CTLs) directed against human CD45. Such CTLs should recognize patient hematopoietic cells including leukemia, enhancing donor cell engraftment and improving the GVL effect, but they should not recognize host nonhematopoietic tissues or donor cells from the graft. Using the T2 binding assay, 4 CD45-derived peptides were found to bind HLA-A2 molecules. These peptides were used to generate cytotoxic T-cell lines from HLA-A2(-) donors by sequential stimulation with peptide-pulsed HLA-A2(+) stimulators, and the lines obtained were screened for peptide-specific cytotoxicity. Using one of these peptides (P1218), it was possible to generate peptide-specific, allorestricted CTLs in 3 of 7 responders. P1218-specific CTL lines show potent cytotoxicity against hematopoietic cell lines coexpressing HLA-A2 and CD45 but not CD45 loss variants. Studies with stable transfectants of 293 cells demonstrated recognition by P1218-specific CTLs of endogenously expressed CD45. Likewise P1218-specific CTLs recognized peripheral blood mononuclear cells (PBMCs) from HLA-A2(+) patients with chronic myeloid leukemia (CML) and leukemic blasts in HLA-A2(+) patients with acute myeloid leukemia (AML), but they were unable to lyse HLA-A2(+) fibroblasts or HLA-A2(-) normal PBMCs. Coculture of CD34(+) PBMCs and bone marrow mononuclear cells (BMMCs) with P1218-specific CTL significantly inhibited colony-forming unit-granulocyte macrophage (CFU-GM) formation in HLA-A2(+) healthy controls and CML patients but resulted in no significant inhibition in HLA-A2(-) healthy controls. These studies demonstrate that P1218-specific cytotoxic T lymphocytes (CTLs) have potent activity against leukemic progenitors and suggest that

  8. Human plasma cells express granzyme B.

    Science.gov (United States)

    Xu, Wei; Narayanan, Priya; Kang, Ning; Clayton, Sandra; Ohne, Yoichiro; Shi, Peiqing; Herve, Marie-Cecile; Balderas, Robert; Picard, Capucine; Casanova, Jean-Laurent; Gorvel, Jean-Pierre; Oh, Sangkon; Pascual, Virginia; Banchereau, Jacques

    2014-01-01

    While studying the plasma cell (PC) compartment in human tonsils, we identified that immunoglobulin kappa or lambda chain-expressing PCs are the main cells expressing granzyme B (GrzB). In vitro studies revealed that activated B cells differentiated into GrzB-expressing PCs when co-cultured with macrophages and follicular helper T cells. This effect could be reproduced on combined stimulation of IL-15 (produced by macrophages) and IL-21 (produced by T follicular helper cells) in a STAT3-dependent manner. Whereas IL-21 triggers the transcription of mRNA of GrzB, IL-15 synergizes the translation of GrzB proteins. The precise role of GrzB in PC biology remains to be understood and studies in mice will not help as their PCs do not express GrzB.

  9. Human CD56bright NK Cells

    DEFF Research Database (Denmark)

    Michel, Tatiana; Poli, Aurélie; Cuapio, Angelica

    2016-01-01

    Human NK cells can be subdivided into various subsets based on the relative expression of CD16 and CD56. In particular, CD56(bright)CD16(-/dim) NK cells are the focus of interest. They are considered efficient cytokine producers endowed with immunoregulatory properties, but they can also become...... cytotoxic upon appropriate activation. These cells were shown to play a role in different disease states, such as cancer, autoimmunity, neuroinflammation, and infection. Although their phenotype and functional properties are well known and have been extensively studied, their lineage relationship with other...... NK cell subsets is not fully defined, nor is their precise hematopoietic origin. In this article, we summarize recent studies about CD56(bright) NK cells in health and disease and briefly discuss the current controversies surrounding them....

  10. Human embryonic stem cells: preclinical perspectives

    Directory of Open Access Journals (Sweden)

    Sarda Kanchan

    2008-01-01

    Full Text Available Abstract Human embryonic stem cells (hESCs have been extensively discussed in public and scientific communities for their potential in treating diseases and injuries. However, not much has been achieved in turning them into safe therapeutic agents. The hurdles in transforming hESCs to therapies start right with the way these cells are derived and maintained in the laboratory, and goes up-to clinical complications related to need for patient specific cell lines, gender specific aspects, age of the cells, and several post transplantation uncertainties. The different types of cells derived through directed differentiation of hESC and used successfully in animal disease and injury models are described briefly. This review gives a brief outlook on the present and the future of hESC based therapies, and talks about the technological advances required for a safe transition from laboratory to clinic.

  11. Preferential response of acute myeloid leukemias with translocation involving chromosome 17 to human recombinant granulocyte colony-stimulating factor.

    Science.gov (United States)

    Pébusque, M J; Lafage, M; Lopez, M; Mannoni, P

    1988-07-01

    Induction of proliferation and differentiation in response to the addition of recombinant human granulocyte colony-stimulating factor (G-CSF) was studied by both suspension and semisolid cultures in a series of acute myeloid leukemias (AML). Induction of proliferation by G-CSF alone was observed in six of 27 cases of AML. All acute promyelocytic leukemias with the specific chromosomal translocation t(15;17) and one case of myelomonocytic leukemia with balanced chromosomal translocation involving chromosome 17 at band q12q21 were induced to proliferate strongly by the G-CSF. However, contrary to the long-term proliferative effect observed with granulocyte/macrophage colony-stimulating factor (GM-CSF), G-CSF activity can be characterized by its capability to initiate and promote the growth of responding AML cells but not to sustain long-term proliferation. Finally, no terminal differentiation was found, as assessed by morphology, cytochemistry, and cell surface marker analysis. These results indicate that G-CSF may be sufficient to provide a specific signal for induction of a transient proliferation in AML without induction of terminal differentiation. The cells with the highest response are clonal leukemia cells, all bearing a translocation involving the chromosome region 17q12q21 in which the G-CSF gene has been recently located.

  12. Stem cell factor and c-Kit in human primordial germ cells and fetal ovaries

    DEFF Research Database (Denmark)

    Høyer, Poul Erik; Byskov, Anne Grete; Møllgård, Kjeld

    2005-01-01

    Prenatal ovary (human), Primordial germ cells, Folliculogenesis, c-Kit, Stem cell factor, immunohistochemistry......Prenatal ovary (human), Primordial germ cells, Folliculogenesis, c-Kit, Stem cell factor, immunohistochemistry...

  13. A Rare Cytogenetic Presentation of Acute Myeloid Leukemia (AML-M2

    Directory of Open Access Journals (Sweden)

    Hanumanthappa Vijay Raghavendra

    2012-12-01

    Full Text Available Acute myeloid leukemia (AML with t(8;21(q22;q22 generating the AML1/ETO fusion gene on 8q22 is a distinct type of AML t(8;21 category (WHO/AML-M2 (FAB, generally associated with a favourable prognosis. Variant additional chromosomal abnormalities are frequently reported. We report three adult cases of this category with unusual karyotype. Bone marrow cytogenetics of case no. 1: 45,X,-Y, t(8;21(q13;q22 with a novel breakpoint of chromosome 8 at (q13. Case no. 2: 46,X,t(X;2(q22;q37,t(3;7(q21;q36,t(5;14(p15;q11,del(8(q22 a complex rearrangement without the involvement of chromosome 21. Case no. 3: 49,XX,+5, t(8;21(q22;q22, +16, +der(21t(8;21(q22;q22 with additional der(21. Endometrial in this case which was positive for myeloperoxidase (MPO and CD117 conforming the AML infiltration. All are morphologically AML with t(8;21. Relevant literature in cytogenetic of AML-M2 is reviewed. The molecular mechanism involved in unusual rearrangements and clinical significance of them are subjected for further studies

  14. Differences in the microrheology of human embryonic stem cells and human induced pluripotent stem cells.

    Science.gov (United States)

    Daniels, Brian R; Hale, Christopher M; Khatau, Shyam B; Kusuma, Sravanti; Dobrowsky, Terrence M; Gerecht, Sharon; Wirtz, Denis

    2010-12-01

    Embryonic and adult fibroblasts can be returned to pluripotency by the expression of reprogramming genes. Multiple lines of evidence suggest that these human induced pluripotent stem (hiPS) cells and human embryonic stem (hES) cells are behaviorally, karyotypically, and morphologically similar. Here we sought to determine whether the physical properties of hiPS cells, including their micromechanical properties, are different from those of hES cells. To this end, we use the method of particle tracking microrheology to compare the viscoelastic properties of the cytoplasm of hES cells, hiPS cells, and the terminally differentiated parental human fibroblasts from which our hiPS cells are derived. Our results indicate that although the cytoplasm of parental fibroblasts is both viscous and elastic, the cytoplasm of hiPS cells does not exhibit any measurable elasticity and is purely viscous over a wide range of timescales. The viscous phenotype of hiPS cells is recapitulated in parental cells with disassembled actin filament network. The cytoplasm of hES cells is predominantly viscous but contains subcellular regions that are also elastic. This study supports the hypothesis that intracellular elasticity correlates with the degree of cellular differentiation and reveals significant differences in the mechanical properties of hiPS cells and hES cells. Because mechanical stimuli have been shown to mediate the precise fate of differentiating stem cells, our results support the concept that stem cell "softness" is a key feature of force-mediated differentiation of stem cells and suggest there may be subtle functional differences between force-mediated differentiation of hiPS cells and hES cells.

  15. Generation of mature hematopoietic cells from human pluripotent stem cells.

    Science.gov (United States)

    Togarrati, Padma Priya; Suknuntha, Kran

    2012-06-01

    A number of malignant and non-malignant hematological disorders are associated with the abnormal production of mature blood cells or primitive hematopoietic precursors. Their capacity for continuous self-renewal without loss of pluripotency and the ability to differentiate into adult cell types from all three primitive germ layers make human embryonic stem cells and induced pluripotent stem cells (hiPSCs) attractive complementary cell sources for large-scale production of transfusable mature blood cell components in cell replacement therapies. The generation of patient-specific hematopoietic stem/precursor cells from iPSCs by the regulated manipulation of various factors involved in reprograming to ensure complete pluripotency, and developing innovative differentiation strategies for generating unlimited supply of clinically safe, transplantable, HLA-matched cells from hiPSCs to outnumber the inadequate source of hematopoietic stem cells obtained from cord blood, bone marrow and peripheral blood, would have a major impact on the field of regenerative and personalized medicine leading to translation of these results from bench to bedside.

  16. Human colostral cells. I. Separation and characterization.

    Science.gov (United States)

    Crago, S S; Prince, S J; Pretlow, T G; McGhee, J R; Mestecky, J

    1979-12-01

    Analyses of the cells present in human colostrum obtained from fifty-four healthy donors during the first four days of lactation revealed that there were 3.3 x 10(6) (range 1.1 x 10(5)--1.2 x 10(7)) cells per ml of colostrum. Based on histochemical examinations, it was found that this population consisted of 30--47% macrophages, 40--60% polymorphonuclear leucocytes, 5.2--8.9% lymphocytes, and 1.3--2.8% colostral corpuscles; epithelial cells were rarely encountered. The identity of various cell types was confirmed by Wright's stain and by a series of histochemical techniques which disclosed the presence of non-specific esterase, peroxidase, and lipids. For further characterization, the different types of cells were separated by various methods, such as Ficoll-Hypaque density centrifugation, isokinetic centrifugation on a linear Ficoll gradient, adherence to glass or plastic, and phagocytosis of carbonyl iron. Immunohistochemical staining with FITC- and/or TRITC-labelled reagents to IgA, IgM, IgG, K- and lambda-chains, secretory component, lactoferrin, and alpha-lactalbumin were applied to unseparated as well as separated colostral cells. Polymorphonuclear leucocytes (staining for peroxidase) as well as macrophages and colostral corpuscles (staining for non-specific esterase) exhibited numerous intracellular vesicles that contained lipids as well as various combinations of milk proteins. Lymphoid cells did not stain with any of these reagents and plasma cells were not detected among the colostral cells. Individual phagocytic cells contained immunoglobulins of the IgA and IgM classes, both K and lambda light chains, secretory component, lactoferrin, and alpha-lactalbumin. The coincidental appearance of these proteins in single, phagocytic cells but not in lymphoid cells indicate that the cells acquired these proteins by ingestion from the environment. Markers commonly used for the identification of B lymphocytes (surface immunoglobulins) and T lymphocytes (receptors

  17. 21 CFR 864.2280 - Cultured animal and human cells.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cultured animal and human cells. 864.2280 Section... (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro...

  18. DNA repair responses in human skin cells

    Energy Technology Data Exchange (ETDEWEB)

    Hanawalt, P.C.; Liu, S.C.; Parsons, C.S.

    1981-07-01

    Sunlight and some environmental chemical agents produce lesions in the DNA of human skin cells that if unrepaired may interfere with normal functioning of these cells. The most serious outcome of such interactions may be malignancy. It is therefore important to develop an understanding of mechanisms by which the lesions may be repaired or tolerated without deleterious consequences. Our models for the molecular processing of damaged DNA have been derived largely from the study of bacterial systems. Some similarities but significant differences are revealed when human cell responses are tested against these models. It is also of importance to learn DNA repair responses of epidermal keratinocytes for comparison with the more extensive studies that have been carried out with dermal fibroblasts. Our experimental results thus far indicate similarities for the excision-repair of ultraviolet-induced pyrimidine dimers in human keratinocytes and fibroblasts. Both the monoadducts and the interstrand crosslinks produced in DNA by photoactivated 8-methoxypsoralen (PUVA) can be repaired in normal human fibroblasts but not in those from xeroderma pigmentosum patients. The monoadducts, like pyrimidine dimers, are probably the more mutagenic/carcinogenic lesions while the crosslinks are less easily repaired and probably result in more effective blocking of DNA function. It is suggested that a split-dose protocol that maximizes the production of crosslinks while minimizing the yield of monoadducts may be more effective and potentially less carcinogenic than the single ultraviolet exposure regimen in PUVA therapy for psoriasis.

  19. Inhibition of the Mitochondrial Protease ClpP as a Therapeutic Strategy for Human Acute Myeloid Leukemia.

    Science.gov (United States)

    Cole, Alicia; Wang, Zezhou; Coyaud, Etienne; Voisin, Veronique; Gronda, Marcela; Jitkova, Yulia; Mattson, Rachel; Hurren, Rose; Babovic, Sonja; Maclean, Neil; Restall, Ian; Wang, Xiaoming; Jeyaraju, Danny V; Sukhai, Mahadeo A; Prabha, Swayam; Bashir, Shaheena; Ramakrishnan, Ashwin; Leung, Elisa; Qia, Yi Hua; Zhang, Nianxian; Combes, Kevin R; Ketela, Troy; Lin, Fengshu; Houry, Walid A; Aman, Ahmed; Al-Awar, Rima; Zheng, Wei; Wienholds, Erno; Xu, Chang Jiang; Dick, John; Wang, Jean C Y; Moffat, Jason; Minden, Mark D; Eaves, Connie J; Bader, Gary D; Hao, Zhenyue; Kornblau, Steven M; Raught, Brian; Schimmer, Aaron D

    2015-06-08

    From an shRNA screen, we identified ClpP as a member of the mitochondrial proteome whose knockdown reduced the viability of K562 leukemic cells. Expression of this mitochondrial protease that has structural similarity to the cytoplasmic proteosome is increased in leukemic cells from approximately half of all patients with AML. Genetic or chemical inhibition of ClpP killed cells from both human AML cell lines and primary samples in which the cells showed elevated ClpP expression but did not affect their normal counterparts. Importantly, Clpp knockout mice were viable with normal hematopoiesis. Mechanistically, we found that ClpP interacts with mitochondrial respiratory chain proteins and metabolic enzymes, and knockdown of ClpP in leukemic cells inhibited oxidative phosphorylation and mitochondrial metabolism.

  20. Inhibition of the mitochondrial protease, ClpP, as a therapeutic strategy for human acute myeloid leuekmia

    Science.gov (United States)

    Cole, Alicia; Wang, Zezhou; Coyaud, Etienne; Voisin, Veronique; Gronda, Marcela; Jitkova, Yulia; Mattson, Rachel; Hurren, Rose; Babovic, Sonja; Maclean, Neil; Restall, Ian; Wang, Xiaoming; Jeyaraju, Danny V.; Sukhai, Mahadeo A.; Prabha, Swayam; Bashir, Shaheena; Ramakrishnan, Ashwin; Leung, Elisa; Qia, Yi Hua; Zhang, Nianxian; Combes, Kevin R.; Ketela, Troy; Lin, Fengshu; Houry, Walid A.; Aman, Ahmed; Al-awar, Rima; Zheng, Wei; Wienholds, Erno; Xu, Chang Jiang; Dick, John; Wang, Jean C.Y.; Moffat, Jason; Minden, Mark D.; Eaves, Connie J.; Bader, Gary D.; Hao, Zhenyue; Kornblau, Steven M.; Raught, Brian; Schimmer, Aaron D.

    2015-01-01

    Summary From an shRNA screen, we identified ClpP as a member of the mitochondrial proteome whose knockdown reduced the viability of K562 leukemic cells. Expression of this mitochondrial protease that has structural similarity to the cytoplasmic proteosome is increased in the leukemic cells from approximately half of patients with AML. Genetic or chemical inhibition of ClpP killed cells from both human AML cell lines and primary samples in which the cells showed elevated ClpP expression, but did not affect their normal counterparts. Importantly, Clpp knockout mice were viable with normal hematopoiesis. Mechanistically, we found ClpP interacts with mitochondrial respiratory chain proteins and metabolic enzymes, and knockdown of ClpP in leukemic cells inhibited oxidative phosphorylation and mitochondrial metabolism. PMID:26058080

  1. Asymptotically Matched Layer (AML) for transient wave propagation in a moving frame of reference

    DEFF Research Database (Denmark)

    Madsen, Stine Skov; Krenk, Steen

    2017-01-01

    The paper presents an Asymptotically Matched Layer (AML) formulation in a moving frame of reference for transient dynamic response of a multi-layer 2D half-space. A displacement based finite element formulation of the convected domain problem is presented together with the AML formulation in which...... the original convolution integrals are represented via two auxiliary displacement-like state-space variables. A parametric study of the AML parameters is conducted for optimizing the absorbing properties. The performance is demonstrated on a single- and a two-layered half-space for various velocities...

  2. Angiomyolipoma (AML) without visible fat: Ultrasound, CT and MR imaging features with pathological correlation

    Energy Technology Data Exchange (ETDEWEB)

    Hakim, Shaheed W.; Flood, Trevor A. [The Ottawa Hospital, University of Ottawa, Department of Anatomical Pathology, Ottawa, Ontario (Canada); Schieda, Nicola; Hodgdon, Taryn; McInnes, Matthew D.F.; Dilauro, Marc [The Ottawa Hospital, University of Ottawa, Department of Medical Imaging, Ottawa, Ontario (Canada)

    2016-02-15

    To compare imaging findings with histopathology in AML without visible fat (AML{sub wvf}). With IRB approval, we identified 18 AML{sub wvf} that underwent CT between 2002-2014. A radiologist measured NECT-attenuation, corticomedullary (CM) and nephrographic (NG) enhancement, echogenicity relative to renal cortex (RC) (N = 5), T2W (T2{sub AML}/T2{sub RC}) signal-intensity (SI), and chemical-shift SI ([SI{sub IN-PHASE} - SI{sub OPPOSED-PHASE}]/SI{sub IN-PHASE}) indices (N = 6). A pathologist re-evaluated 15/18 AML{sub wvf} for 1) < or > 25 % adipocytes/high-power-field (HPF), 2) ''many or few'' blood vessels. Comparisons were performed using chi-square and independent t-tests. 73.3 %(11/15) of AML{sub wvf} had <25 % adipocytes/HPF and 86.7 %(13/15) had ''many'' blood vessels. NECT-attenuation was 41.8(±6.9) HU. 61.1 %(11/18) of AML{sub wvf} were hyper-attenuating and 38.9 %(7/18) iso-attenuating; attenuation was associated with %-adipocytes/HPF, (p = 0.01). CM/NG enhancement were 63.3(±20.8)/51.7(±15.5) HU. 72.2 %(13/18) of AML{sub wvf} had wash-out enhancement, with no association with amount of blood vessels at pathology, (p = 0.68). No difference in echogenicity was noted by histology (p > 0.05). All AML{sub wvf} were T2-hypointense (SI ratio = 0.61 [±0.1]). 2/6 AML{sub wvf} showed SI drop on chemical-shift MRI; both were iso-attenuating and were associated with >25 % adipocytes/HPF (p = 0.04). AML{sub wvf} are typically T2-hypointense and hyper-attenuating with wash-out enhancement due to abundant smooth muscle and vessels respectively. Iso-attenuating AML{sub wvf} with microscopic fat on MRI contain more adipocytes/HPF. (orig.)

  3. Identification of novel genomic aberrations in AML-M5 in a level of array CGH.

    Directory of Open Access Journals (Sweden)

    Rui Zhang

    Full Text Available To assess the possible existence of unbalanced chromosomal abnormalities and delineate the characterization of copy number alterations (CNAs of acute myeloid leukemia-M5 (AML-M5, R-banding karyotype, oligonucelotide array CGH and FISH were performed in 24 patients with AML-M5. A total of 117 CNAs with size ranging from 0.004 to 146.263 Mb was recognized in 12 of 24 cases, involving all chromosomes other than chromosome 1, 4, X and Y. Cryptic CNAs with size less than 5 Mb accounted for 59.8% of all the CNAs. 12 recurrent chromosomal alterations were mapped. Seven out of them were described in the previous AML studies and five were new candidate AML-M5 associated CNAs, including gains of 3q26.2-qter and 13q31.3 as well as losses of 2q24.2, 8p12 and 14q32. Amplication of 3q26.2-qter was the sole large recurrent chromosomal anomaly and the pathogenic mechanism in AML-M5 was possibly different from the classical recurrent 3q21q26 abnormality in AML. As a tumor suppressor gene, FOXN3, was singled out from the small recurrent CNA of 14q32, however, it is proved that deletion of FOXN3 is a common marker of myeloid leukemia rather than a specific marker for AML-M5 subtype. Moreover, the concurrent amplication of MLL and deletion of CDKN2A were noted and it might be associated with AML-M5. The number of CNA did not show a significant association with clinico-biological parameters and CR number of the 22 patients received chemotherapy. This study provided the evidence that array CGH served as a complementary platform for routine cytogenetic analysis to identify those cryptic alterations in the patients with AML-M5. As a subtype of AML, AML-M5 carries both common recurrent CNAs and unique CNAs, which may harbor novel oncogenes or tumor suppressor genes. Clarifying the role of these genes will contribute to the understanding of leukemogenic network of AML-M5.

  4. High EVI1 expression predicts poor survival in acute myeloid leukemia: a study of 319 de novo AML patients.

    Science.gov (United States)

    Barjesteh van Waalwijk van Doorn-Khosrovani, Sahar; Erpelinck, Claudia; van Putten, Wim L J; Valk, Peter J M; van der Poel-van de Luytgaarde, Sonja; Hack, Ronald; Slater, Rosalyn; Smit, Elisabeth M E; Beverloo, H Berna; Verhoef, Gregor; Verdonck, Leo F; Ossenkoppele, Gert J; Sonneveld, Pieter; de Greef, Georgine E; Löwenberg, Bob; Delwel, Ruud

    2003-02-01

    The proto-oncogene EVI1 encodes a DNA binding protein and is located on chromosome 3q26. The gene is aberrantly expressed in acute myeloid leukemia (AML) patients carrying 3q26 abnormalities. Two mRNAs are transcribed from this locus: EVI1 and a fusion of EVI1 with MDS1 (MDS1-EVI1), a gene located 5' of EVI1. The purpose of this study was to investigate which of the 2 gene products is involved in transformation in human AML. To discriminate between EVI1 and MDS1-EVI1 transcripts, distinct real-time quantitative polymerase chain reaction (PCR) assays were developed. Patients with 3q26 abnormalities often showed high EVI1 and MDS1-EVI1 expression. In a cohort of 319 AML patients, 4 subgroups could be distinguished: EVI1(+) and MDS1-EVI1(-) (6 patients; group I), EVI1(+) and MDS1-EVI1(+) (26 patients; group II), EVI1(-) and MDS1-EVI1(+) (12 patients; group III), and EVI1(-) and MDS1-EVI1(-) (275 patients; group IV). The only 4 patients with a 3q26 aberration belonged to groups I and II. Interestingly, high EVI1 and not MDS1-EVI1 expression was associated with unfavorable karyotypes (eg, -7/7q-) or complex karyotypes. Moreover, a significant correlation was observed between EVI1 expression and 11q23 aberrations (mixed lineage leukemia [MLL] gene involvement). Patients from groups I and II had significantly shorter overall and event-free survival than patients in groups III and IV. Our data demonstrate that high EVI1 expression is an independent poor prognostic marker within the intermediate- risk karyotypic group.

  5. Human T Cell Memory: A Dynamic View

    Directory of Open Access Journals (Sweden)

    Derek C. Macallan

    2017-02-01

    Full Text Available Long-term T cell-mediated protection depends upon the formation of a pool of memory cells to protect against future pathogen challenge. In this review we argue that looking at T cell memory from a dynamic viewpoint can help in understanding how memory populations are maintained following pathogen exposure or vaccination. For example, a dynamic view resolves the apparent paradox between the relatively short lifespans of individual memory cells and very long-lived immunological memory by focussing on the persistence of clonal populations, rather than individual cells. Clonal survival is achieved by balancing proliferation, death and differentiation rates within and between identifiable phenotypic pools; such pools correspond broadly to sequential stages in the linear differentiation pathway. Each pool has its own characteristic kinetics, but only when considered as a population; single cells exhibit considerable heterogeneity. In humans, we tend to concentrate on circulating cells, but memory T cells in non-lymphoid tissues and bone marrow are increasingly recognised as critical for immune defence; their kinetics, however, remain largely unexplored. Considering vaccination from this viewpoint shifts the focus from the size of the primary response to the survival of the clone and enables identification of critical system pinch-points and opportunities to improve vaccine efficacy.

  6. Biological impact of human embryonic stem cells.

    Science.gov (United States)

    Martín, Miguel; Menéndez, Pablo

    2012-01-01

    Research on human embryonic stem cells (hESCs) and induced pluripotent (iPS) stem cells is currently a field of great potential in biomedicine. These cells represent a highly valuable tool for developmental biology studies, disease models, and drug screening and toxicity. The ultimate goal of hESCs and iPS cell research is the treatment of diseases or disorders for which there is currently no treatment or existing therapies are only partially effective. Despite the disproportionate short-term hopes generated, which are putting too much pressure on scientists, the international scientific community is making rapid progress in understanding hESCs and iPS cells. Nonetheless, great efforts have to be made to provide an answer to still quite basic questions concerning their biology. Moreover, translation to clinical applications in cell replacement therapy requires prior solution to ethical barriers. The recent development of iPS cells has provided a strong alternative to overcome ethical issues concerning hESCs. However, an in-depth characterization of their genetic and epigenetic features, as well as their differentiation potential still remains to be undertaken. This chapter will describe, precisely, what the critical issues are, where scientific and ethical barriers stand, and how we are to overcome them. Only then, we shall finally discover whether hESCs and iPS cells will allow building reproducible disease models, and whether they really are a safe tool, with great potential for regenerative medicine.

  7. Role for c-jun N-terminal kinase in treatment-refractory acute myeloid leukemia (AML): signaling to multidrug-efflux and hyperproliferation.

    Science.gov (United States)

    Cripe, L D; Gelfanov, V M; Smith, E A; Spigel, D R; Phillips, C A; Gabig, T G; Jung, S-H; Fyffe, J; Hartman, A D; Kneebone, P; Mercola, D; Burgess, G S; Boswell, H S

    2002-05-01

    A relationship was proved between constitutive activity of leukemic cell c-jun-N-terminal kinase (JNK) and treatment failure in AML. Specifically, early treatment failure was predicted by the presence of constitutive JNK activity. The mechanistic origins of this association was sought. A multidrug resistant leukemic cell line, HL-60/ADR, characterized by hyperexpression of c-jun and JNK activity, was transfected with a mutant c-jun vector, whose substrate N-terminal c-jun serines were mutated. Down-regulated expression occurred of c-jun/AP-1-dependent genes, catalase and glutathione-S-transferase (GST) pi, which participate in cellular homeostasis to oxidative stress and xenobiotic exposure. MRP-efflux was abrogated in HL-60/ADR cells with dominant-negative c-jun, perhaps because MRP1 protein expression was also lost. Heightened sensitivity to daunorubicin resulted in cells subjected to this change. Biochemical analysis in 67 primary adult AML samples established a statistical correlation between cellular expression of c-jun and JNK activity, JNK activity with hyperleukocytosis at presentation of disease, and with exuberant MRP efflux. These findings reflect the survival role for c-jun/AP-1 and its regulatory kinase previously demonstrated for yeast in homeostatic response to oxidative stress and in operation of ATP-binding cassette efflux pumps, and may support evolutionary conservation of such function. Thus, JNK and c-jun may be salient drug targets in multidrug resistant AML.

  8. Human fetal liver stromal cells expressing erythropoietin promote hematopoietic development from human embryonic stem cells.

    Science.gov (United States)

    Yang, Chao; Ji, Lei; Yue, Wen; Shi, Shuang-Shuang; Wang, Ruo-Yong; Li, Yan-Hua; Xie, Xiao-Yan; Xi, Jia-Fei; He, Li-Juan; Nan, Xue; Pei, Xue-Tao

    2012-02-01

    Blood cells transfusion and hematopoietic stem cells (HSCs) transplantation are important methods for cell therapy. They are widely used in the treatment of incurable hematological disorder, infectious diseases, genetic diseases, and immunologic deficiency. However, their availability is limited by quantity, capacity of proliferation and the risk of blood transfusion complications. Recently, human embryonic stem cells (hESCs) have been shown to be an alternative resource for the generation of hematopoietic cells. In the current study, we describe a novel method for the efficient production of hematopoietic cells from hESCs. The stable human fetal liver stromal cell lines (hFLSCs) expressing erythropoietin (EPO) were established using the lentiviral system. We observed that the supernatant from the EPO transfected hFLSCs could induce the hESCs differentiation into hematopoietic cells, especially erythroid cells. They not only expressed fetal and embryonic globins but also expressed the adult-globin chain on further maturation. In addition, these hESCs-derived erythroid cells possess oxygen-transporting capacity, which indicated hESCs could generate terminally mature progenies. This should be useful for ultimately developing an animal-free culture system to generate large numbers of erythroid cells from hESCs and provide an experimental model to study early human erythropoiesis.

  9. In vitro priming of tumor-specific cytotoxic T lymphocytes using allogeneic dendritic cells derived from the human MUTZ-3 cell line.

    Science.gov (United States)

    Santegoets, Saskia J A M; Schreurs, Marco W J; Masterson, Allan J; Liu, Ying Poi; Goletz, Steffen; Baumeister, Hans; Kueter, Esther W M; Lougheed, Sinéad M; van den Eertwegh, Alfons J M; Scheper, Rik J; Hooijberg, Erik; de Gruijl, Tanja D

    2006-12-01

    The adoptive transfer of in vitro-induced and expanded tumor-specific cytotoxic T lymphocytes (CTL) presents a promising immunotherapeutic approach for the treatment of cancer. The in vitro induction of tumor-reactive CTL requires repeated stimulation of CTL precursors with dendritic cells (DC). To circumvent problems like scarcity of blood DC precursors and donor variability, it would be attractive to use DC from a non-autologous, unlimited source. DCs derived from the human acute myeloid leukemia (AML) cell line MUTZ-3 are attractive candidates since these DCs closely resemble monocyte-derived DC (MoDC) in terms of phenotype and T cell stimulatory capacity. Here we demonstrate that functional CTL clones could be generated against multiple tumor-associated antigens, i.e., human telomerase reverse transcriptase (hTERT), ErbB3-binding protein-1 (Ebp1), carcinoembryonic antigen (CEA) and Her-2/neu, by stimulating CD8beta(+) CTL precursors with peptide-loaded allogeneic, HLA-A2-matched MUTZ-3-derived DC. A consistent induction capacity, as determined by MHC tetramer-binding, was found in multiple donors and comparable to autologous peptide-loaded MoDC. Functional characterization at the clonal level revealed the priming of CTL that recognized endogenously processed epitopes on tumor cell lines in an HLA-A2-restricted fashion. Our data indicate that MUTZ-3-derived DC can be used as stimulator cells for in vitro priming and expansion of functional TAA-specific effector CTL. MUTZ-3-derived DCs thus represent a ready and standardized source of allogeneic DC to generate CTL for therapeutic adoptive transfer strategies.

  10. Core binding factor acute myeloid leukemia (CBF-AML) in México: a single institution experience.

    Science.gov (United States)

    Ruiz-Delgado, Guillermo J; Macías-Gallardo, Julio; Lutz-Presno, Julia; Garcés-Eisele, Javier; Hernández-Arizpe, Ana; Montes-Montiel, Maryel; Ruiz-Argüelles, Guillermo J

    2011-01-01

    Twenty one patients with CBF-AML presented prospectively in the Centro de Hematología y Medicina Interna de Puebla (Puebla, México) between February 1995 and March 2010, 14 with the t(8;21)(q22;q22) and 7 with the inv(16)(p13;q22)/t(16;16)(p13;q22); they represent 13% of all cases of AML. The median age of the patients was 24 years (range 1 to 61). Seven of 14 patients with t(8;21)(q22;q22) had an M2 morphology whereas 3/7 with the inv(16) had an M4 morphology; in addition to the myeloid markers identified by flow-cytometry (surface CD13, surface CD33, and cytoplasmic myeloperoxidase) lymphoid markers were identified in the blast cells of 8/14 cases of the t(8;21) patients, but in no patient with the inv(16). Nineteen patients were treated with combined chemotherapy and 16 (84%) achieved a complete molecular remission. Seven patients were auto or allografted. Relapses presented in 10/16 patients. The median probability of overall survival (OS) has not been reached being above 165 months, whereas the 165-month probability of OS and leukemia-free survival was 52%; despite a tendency for a better outcome of patients with the t(8;21), there were no significant differences in survival of patients with either the t(8;21) or the inv(16). In this single institution experience in México, we found that the CBF variants of AML have a similar prevalence as compared with Caucasian populations, that the co-expression of lymphoid markers in the blast cells was frequent in the t(8;21) and that these two AML subtypes were associated with a relatively good long-term prognosis. Further studies are needed to describe with more detail the precise biological features of these molecular subtypes of acute leukemia.

  11. Cell phoney: human cloning after Quintavalle.

    Science.gov (United States)

    Morgan, Derek; Ford, Mary

    2004-12-01

    Reproductive cloning has thrown up new scientific possibilities, ethical conundrums, and legal challenges. An initial question, considered by the English courts in 2003, was whether the technique presently available, that of cell nucleus replacement, falls outside the provisions of the Human Fertilisation and Embryology Act 1990. If it does, the creation and use, including use in research protocols, of human embryos would be unregulated, disclosing a need to consider remedial legislation. The resolution by the courts of this legal question dramatically engages them in a resolution of fundamental ethical dilemmas, and discloses the possibilities and limitation of negotiating science policy through the processes of litigation.

  12. Lymphoid Cell-Glioma Cell Interaction Enhances Cell Coat Production by Human Gliomas: Novel Suppressor Mechanism

    Science.gov (United States)

    Dick, Steven J.; Macchi, Beatrice; Papazoglou, Savvas; Oldfield, Edward H.; Kornblith, Paul L.; Smith, Barry H.; Gately, Maurice K.

    1983-05-01

    Certain human glioma lines produce mucopolysaccharide coats that impair the generation of cytolytic lymphocytes in response to these lines in vitro. Coat production is substantially enhanced by the interaction of glioma cells with a macromolecular factor released by human peripheral blood mononuclear cells in culture. This interaction thus constitutes an unusual mechanism by which inflammatory cells may nonspecifically suppress the cellular immune response to at least one class of solid tumors in humans.

  13. Human induced pluripotent stem cell-derived models to investigate human cytomegalovirus infection in neural cells.

    Directory of Open Access Journals (Sweden)

    Leonardo D'Aiuto

    Full Text Available Human cytomegalovirus (HCMV infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs, neural progenitor cells (NPCs and neurons suggests that (i iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii Neural stem cells have impaired differentiation when infected by HCMV; (iii NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv most iPS-derived neurons are not permissive to HCMV infection; and (v infected neurons have impaired calcium influx in response to glutamate.

  14. Impact of cytogenetics on outcome of de novo and therapy-related AML and MDS after allogeneic transplantation.

    Science.gov (United States)

    Armand, Philippe; Kim, Haesook T; DeAngelo, Daniel J; Ho, Vincent T; Cutler, Corey S; Stone, Richard M; Ritz, Jerome; Alyea, Edwin P; Antin, Joseph H; Soiffer, Robert J

    2007-06-01

    Cytogenetics has an important impact on the prognosis of patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) for acute myelogenous leukemia (AML) or myelodysplastic syndromes (MDS). However, it is unclear whether currently accepted cytogenetic risk groups, which were established for patients treated mostly with standard therapy, are optimally discriminating for patients undergoing HSCT. Also, the impact of cytogenetics in the growing population of patients with therapy-related disease has not been completely elucidated. In this study, we retrospectively analyzed data on 556 patients with AML or MDS transplanted at our institution. We examined, in multivariate analyses, the contribution of cytogenetics to survival, relapse, and nonrelapse mortality for the 476 patients with de novo disease. We used these results to establish an optimal cytogenetic grouping scheme. We then applied this grouping scheme to the 80 patients with therapy-related disease. Our proposed 3-group cytogenetic classification outperformed the established grouping schemes for both de novo and therapy-related disease. When classified by this new scheme, cytogenetics was the strongest prognostic factor for overall survival in our cohort, through its impact on the risk of relapse (and not on nonrelapse mortality). After accounting for cytogenetics, patients with therapy-related AML or MDS had an equivalent outcome to those with de novo disease. This study demonstrates the impact of cytogenetics on the risk of relapse and death for patients with both de novo and therapy-related disease undergoing transplantation; it also emphasizes the necessity of using cytogenetics to stratify patients entering clinical trials, and provides a system for doing so, which can be validated in a multi-institutional database.

  15. Human somatic cell nuclear transfer and cloning.

    Science.gov (United States)

    2012-10-01

    This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer (cloning)," last published in Fertil Steril 2000;74:873-6. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  16. Epinecidin-1, an antimicrobial peptide from fish (Epinephelus coioides) which has an antitumor effect like lytic peptides in human fibrosarcoma cells.

    Science.gov (United States)

    Lin, Wei-Ju; Chien, Yi-Lun; Pan, Chia-Yu; Lin, Tai-Lang; Chen, Jyh-Yih; Chiu, Shu-Jun; Hui, Cho-Fat

    2009-02-01

    Epinecidin-1, a synthetic 21-mer antimicrobial peptide originally identified from grouper (Epinephelus coioides), specifically exhibited high antimicrobial activities against both Gram-negative and Gram-positive bacteria. In the current study we report on the in vitro cytotoxicity of the peptide, an important factor before it can be considered for further applications in cancer therapy. The cytotoxicity of epinecidin-1 was investigated against several cancer cells (A549, HA59T/VGH, HeLa, HepG2, HT1080, RAW264.7, and U937) and normal cells (AML-12, NIH3T3, and WS-1) with the MTT assay, and the inhibition of cancer cell growth was confirmed by a soft agar assay and scanning electron microscopy. However, cell variations were detected with AO/EtBr staining, while apoptosis and necrosis gene expressions in HT1080 cells after treatment with the epinecidin-1 peptide and Nec-1 showed that epinecidin-1 had an anti-necrosis function in HT1080 cells. The data presented here indicate that epinecidin-1 has in vitro antitumor activity against the HT1080 cell line, and functions like lytic peptides. In addition, our results suggest that epinecidin-1 may prove to be an effective chemotherapeutic agent for human fibrosarcoma cells in the future.

  17. Dopamine receptor repertoire of human granulosa cells

    Directory of Open Access Journals (Sweden)

    Kunz Lars

    2007-10-01

    Full Text Available Abstract Background High levels of dopamine (DA were described in human ovary and recently evidence for DA receptors in granulosa and luteal cells has been provided, as well. However, neither the full repertoire of ovarian receptors for DA, nor their specific role, is established. Human granulosa cells (GCs derived from women undergoing in vitro fertilization (IVF are an adequate model for endocrine cells of the follicle and the corpus luteum and were therefore employed in an attempt to decipher their DA receptor repertoire and functionality. Methods Cells were obtained from patients undergoing IVF and examined using cDNA-array, RT-PCR, Western blotting and immunocytochemistry. In addition, calcium measurements (with FLUO-4 were employed. Expression of two DA receptors was also examined by in-situ hybridization in rat ovary. Effects of DA on cell viability and cell volume were studied by using an ATP assay and an electronic cell counter system. Results We found members of the two DA receptor families (D1- and D2 -like associated with different signaling pathways in human GCs, namely D1 (as expected and D5 (both are Gs coupled and linked to cAMP increase and D2, D4 (Gi/Gq coupled and linked to IP3/DAG. D3 was not found. The presence of the trophic hormone hCG (10 IU/ml in the culture medium for several days did not alter mRNA (semiquantitative RT-PCR or protein levels (immunocytochemistry/Western blotting of D1,2,4,5 DA receptors. Expression of prototype receptors for the two families, D1 and D2, was furthermore shown in rat granulosa and luteal cells by in situ hybridization. Among the DA receptors found in human GCs, D2 expression was marked both at mRNA and protein levels and it was therefore further studied. Results of additional RT-PCR and Western blots showed two splice variants (D2L, D2S. Irrespective of these variants, D2 proved to be functional, as DA raised intracellular calcium levels. This calcium mobilizing effect of DA was observed

  18. TALEN-Induced Translocations in Human Cells.

    Science.gov (United States)

    Piganeau, Marion; Renouf, Benjamin; Ghezraoui, Hind; Brunet, Erika

    2016-01-01

    Induction of chromosomal translocations in human cells is of a great interest to study tumorigenesis and genome instability. Here, we explain in detail a method to induce translocations using the transcription activator-like effector nucleases (TALENs). We describe how to detect translocation formation by PCR, calculate translocation frequency by 96-well PCR screen, and analyze breakpoint junctions. When inducing cancer translocations, it is also possible to detect the fusion gene by FISH analysis or western blot.

  19. Azacitidine for treatment of imminent relapse in MDS or AML patients after allogeneic HSCT: results of the RELAZA trial

    Science.gov (United States)

    Platzbecker, U; Wermke, M; Radke, J; Oelschlaegel, U; Seltmann, F; Kiani, A; Klut, I-M; Knoth, H; Röllig, C; Schetelig, J; Mohr, B; Graehlert, X; Ehninger, G; Bornhäuser, M; Thiede, C

    2012-01-01

    This study evaluated azacitidine as treatment of minimal residual disease (MRD) determined by a sensitive donor chimerism analysis of CD34+ blood cells to pre-empt relapse in patients with CD34+ myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (HSCT). At a median of 169 days after HSCT, 20/59 prospectively screened patients experienced a decrease of CD34+ donor chimerism to <80% and received four azacitidine cycles (75 mg/m2/day for 7 days) while in complete hematologic remission. A total of 16 patients (80%) responded with either increasing CD34+ donor chimerism to ⩾80% (n=10; 50%) or stabilization (n=6; 30%) in the absence of relapse. Stabilized patients and those with a later drop of CD34+ donor chimerism to <80% after initial response were eligible for subsequent azacitidine cycles. A total of 11 patients (55%) received a median of 4 (range, 1–11) additional cycles. Eventually, hematologic relapse occurred in 13 patients (65%), but was delayed until a median of 231 days (range, 56–558) after initial decrease of CD34+ donor chimerism to <80%. In conclusion, pre-emptive azacitidine treatment has an acceptable safety profile and can substantially prevent or delay hematologic relapse in patients with MDS or AML and MRD after allogeneic HSCT. PMID:21886171

  20. Comparing cancer vs normal gene expression profiles identifies new disease entities and common transcriptional programs in AML patients

    DEFF Research Database (Denmark)

    Rapin, Nicolas; Bagger, Frederik Otzen; Jendholm, Johan

    2014-01-01

    Gene expression profiling has been used extensively to characterize cancer, identify novel subtypes, and improve patient stratification. However, it has largely failed to identify transcriptional programs that differ between cancer and corresponding normal cells and has not been efficient in iden......-karyotype AML, which allowed for the generation of a highly prognostic survival signature. Collectively, our CvN method holds great potential as a tool for the analysis of gene expression profiles of cancer patients.......Gene expression profiling has been used extensively to characterize cancer, identify novel subtypes, and improve patient stratification. However, it has largely failed to identify transcriptional programs that differ between cancer and corresponding normal cells and has not been efficient...... in identifying expression changes fundamental to disease etiology. Here we present a method that facilitates the comparison of any cancer sample to its nearest normal cellular counterpart, using acute myeloid leukemia (AML) as a model. We first generated a gene expression-based landscape of the normal...

  1. The core regulatory network in human cells.

    Science.gov (United States)

    Kim, Man-Sun; Kim, Dongsan; Kang, Nam Sook; Kim, Jeong-Rae

    2017-03-04

    In order to discover the common characteristics of various cell types in the human body, many researches have been conducted to find the set of genes commonly expressed in various cell types and tissues. However, the functional characteristics of a cell is determined by the complex regulatory relationships among the genes rather than by expressed genes themselves. Therefore, it is more important to identify and analyze a core regulatory network where all regulatory relationship between genes are active across all cell types to uncover the common features of various cell types. Here, based on hundreds of tissue-specific gene regulatory networks constructed by recent genome-wide experimental data, we constructed the core regulatory network. Interestingly, we found that the core regulatory network is organized by simple cascade and has few complex regulations such as feedback or feed-forward loops. Moreover, we discovered that the regulatory links from genes in the core regulatory network to genes in the peripheral regulatory network are much more abundant than the reverse direction links. These results suggest that the core regulatory network locates at the top of regulatory network and plays a role as a 'hub' in terms of information flow, and the information that is common to all cells can be modified to achieve the tissue-specific characteristics through various types of feedback and feed-forward loops in the peripheral regulatory networks. We also found that the genes in the core regulatory network are evolutionary conserved, essential and non-disease, non-druggable genes compared to the peripheral genes. Overall, our study provides an insight into how all human cells share a common function and generate tissue-specific functional traits by transmitting and processing information through regulatory network.

  2. The effect of oleuropein from olive leaf (Olea europaea) extract on Ca²⁺ homeostasis, cytotoxicity, cell cycle distribution and ROS signaling in HepG2 human hepatoma cells.

    Science.gov (United States)

    Cheng, Jin-Shiung; Chou, Chiang-Ting; Liu, Yuan-Yuarn; Sun, Wei-Chih; Shieh, Pochuen; Kuo, Daih-Huang; Kuo, Chun-Chi; Jan, Chung-Ren; Liang, Wei-Zhe

    2016-05-01

    Oleuropein, a phenolic compound found in the olive leaf (Olea europaea), has been shown to have biological activities in different models. However, the effects of oleuropein on Ca(2+) homeostasis, cytotoxicity, cell cycle distribution and ROS signaling in liver cells have not been analyzed. Oleuropein induced [Ca(2+)]i rises only in HepG2 cells but not in AML12, HA22T or HA59T cells due to the different status of 3-hydroxy-3-methylglutaryl-CoA reductase expression. In HepG2 cells, this Ca(2+) signaling response was reduced by removing extracellular Ca(2+), and was inhibited by the store-operated Ca(2+) channel blockers 2-APB and SKF96365. In Ca(2+)-free medium, pretreatment with the ER Ca(2+) pump inhibitor thapsigargin abolished oleuropein-induced [Ca(2+)]i rises. Oleuropein induced cell cycle arrest which was associated with the regulation of p53, p21, CDK1 and cyclin B1 levels. Furthermore, oleuropein elevated intracellular ROS levels but reduced GSH levels. Treatment with the intracellular Ca(2+) chelator BAPTA-AM or the antioxidant NAC partially reversed oleuropein-induced cytotoxicity. Together, in HepG2 cells, oleuropein induced [Ca(2+)]i rises by releasing Ca(2+) from the ER and causing Ca(2+) influx through store-operated Ca(2+) channels. Moreover, oleuropein induced Ca(2+)-associated cytotoxicity that involved ROS signaling and cell cycle arrest. This compound may offer a potential therapy for treatment of human hepatoma.

  3. Cell Culture Assay for Human Noroviruses [response

    Energy Technology Data Exchange (ETDEWEB)

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.

    2007-07-01

    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  4. Preliminary study on human fibroblasts as feeder layer for human embryonic stem cells culture in vitro

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    To avoid the direct contact with mouse cells and possible heterogeneous pathogen in future application, we need to replace mouse embryonic fibroblastswith human fibroblasts as the feeder layer to maintain human embryonic stem cells growth in the undifferentiated state. We successfully use human fibroblasts derived from aborted fetus and adult prepuce as feeder layer to maintain human embryonic stem cells growth. During the passage and growth on this feeder layer, the human embryonic stem cells can keep their undifferentiated state.

  5. Timothy Ley, M.D., Advocates for Personalized Medicine in AML - TCGA

    Science.gov (United States)

    Oncologist Dr. Timothy Ley talks about how repurposing of existing drugs based on better understanding of the genetic basis of acute myeloid leukemia (AML) can help patients receive personalized care.

  6. Primitive cardiac cells from human embryonic stem cells.

    Science.gov (United States)

    Hudson, James; Titmarsh, Drew; Hidalgo, Alejandro; Wolvetang, Ernst; Cooper-White, Justin

    2012-06-10

    Pluripotent stem cell-derived cardiomyocytes are currently being investigated for in vitro human heart models and as potential therapeutics for heart failure. In this study, we have developed a differentiation protocol that minimizes the need for specific human embryonic stem cell (hESC) line optimization. We first reduced the heterogeneity that exists within the starting population of bulk cultured hESCs by using cells adapted to single-cell passaging in a 2-dimensional (2D) culture format. Compared with bulk cultures, single-cell cultures comprised larger fractions of TG30(hi)/OCT4(hi) cells, corresponding to an increased expression of pluripotency markers OCT4 and NANOG, and reduced expression of early lineage-specific markers. A 2D temporal differentiation protocol was then developed, aimed at reducing the inherent heterogeneity and variability of embryoid body-based protocols, with induction of primitive streak cells using bone morphogenetic protein 4 and activin A, followed by cardiogenesis via inhibition of Wnt signaling using the small molecules IWP-4 or IWR-1. IWP-4 treatment resulted in a large percentage of cells expressing low amounts of cardiac myosin heavy chain and expression of early cardiac progenitor markers ISL1 and NKX2-5, thus indicating the production of large numbers of immature cardiomyocytes (~65,000/cm(2) or ~1.5 per input hESC). This protocol was shown to be effective in HES3, H9, and, to a lesser, extent, MEL1 hESC lines. In addition, we observed that IWR-1 induced predominantly atrial myosin light chain (MLC2a) expression, whereas IWP-4 induced expression of both atrial (MLC2a) and ventricular (MLC2v) forms. The intrinsic flexibility and scalability of this 2D protocol mean that the output population of primitive cardiomyocytes will be particularly accessible and useful for the investigation of molecular mechanisms driving terminal cardiomyocyte differentiation, and potentially for the future treatment of heart failure.

  7. Appearance of Human Plasma Cells Following Differentiation of Human B Cells in NOD/SCID Mouse Spleen

    OpenAIRE

    2003-01-01

    Relatively little is known for the differentiation and maturation process of human B cells to plasma cells. This is particularly important in reconstitution work involving transfer of autoantibodies. To address this issue, we transplanted human peripheral blood mononuclear cells (PBMC) directly into the spleen of irradiated NOD/SCID mice depleted of natural killer cell activity. Within 6 weeks, naïve B cells differentiated into memory B cells and, importantly, the numbers of human CD138+ plas...

  8. Establishment of Human Neural Progenitor Cells from Human Induced Pluripotent Stem Cells with Diverse Tissue Origins

    Directory of Open Access Journals (Sweden)

    Hayato Fukusumi

    2016-01-01

    Full Text Available Human neural progenitor cells (hNPCs have previously been generated from limited numbers of human induced pluripotent stem cell (hiPSC clones. Here, 21 hiPSC clones derived from human dermal fibroblasts, cord blood cells, and peripheral blood mononuclear cells were differentiated using two neural induction methods, an embryoid body (EB formation-based method and an EB formation method using dual SMAD inhibitors (dSMADi. Our results showed that expandable hNPCs could be generated from hiPSC clones with diverse somatic tissue origins. The established hNPCs exhibited a mid/hindbrain-type neural identity and uniform expression of neural progenitor genes.

  9. Human microglial cells synthesize albumin in brain.

    Directory of Open Access Journals (Sweden)

    Sung-Min Ahn

    Full Text Available Albumin, an abundant plasma protein with multifunctional properties, is mainly synthesized in the liver. Albumin has been implicated in Alzheimer's disease (AD since it can bind to and transport amyloid beta (Abeta, the causative agent of AD; albumin is also a potent inhibitor of Abeta polymerization. Despite evidence of non-hepatic transcription of albumin in many tissues including kidney and pancreas, non-hepatic synthesis of albumin at the protein level has been rarely confirmed. In a pilot phase study of Human Brain Proteome Project, we found evidence that microglial cells in brain may synthesize albumin. Here we report, for the first time, the de novo synthesis of albumin in human microglial cells in brain. Furthermore, we demonstrate that the synthesis and secretion of albumin from microglial cells is enhanced upon microglial activation by Abeta(1-42- or lipopolysaccharide (LPS-treatment. These data indicate that microglial cells may play a beneficial role in AD by secreting albumin that not only inhibits Abeta polymerization but also increases its clearance.

  10. In acute myeloid leukemia, B7-H1 (PD-L1) protection of blasts from cytotoxic T cells is induced by TLR ligands and interferon-gamma and can be reversed using MEK inhibitors.

    Science.gov (United States)

    Berthon, Céline; Driss, Virginie; Liu, Jizhong; Kuranda, Klaudia; Leleu, Xavier; Jouy, Nathalie; Hetuin, Dominique; Quesnel, Bruno

    2010-12-01

    B7-H1 (PD-L1) is a B7-related protein that inhibits T-cell responses. B7-H1 participates in the immunoescape of cancer cells and is also involved in the long-term persistence of leukemic cells in a mouse model of leukemia. B7-H1 can be constitutively expressed by cancer cells, but is also induced by various stimuli. Therefore, we examined the constitutive and inducible expression of B7-H1 and the consequences of this expression in human acute myeloid leukemia (AML). We analyzed B7-H1 expression in a cohort of 79 patients with AML. In addition, we studied blast cells after incubation with interferon-gamma or toll-like receptors (TLR) ligands. Finally, we evaluated functionality of cytotoxic T-cell activity against blast cells. Expression of B7-H1 upon diagnosis was high in 18% of patients. Expression of TLR2, 4 and 9 was detected in one-third of AML samples. Expression of TLR2 and TLR4 ligands or IFN-γ induced by B7-H1 was found to protect AML cells from CTL-mediated lysis. Spontaneous B7-H1 expression was also found to be enhanced upon relapse in some patients. MEK inhibitors, including UO126 and AZD6244, reduced B7-H1 expression and restored CTL-mediated lysis of blast cells. In AML, B7-H1 expression by blasts represents a possible immune escape mechanism. The inducibility of B7-H1 expression by IFN-γ or TLR ligands suggests that various stimuli, either produced during the immune response against leukemia cells or released by infectious microorganisms, could protect leukemic cells from T cells. The efficacy of MEK inhibitors against B7-H1-mediated inhibition of CTLs suggests a possible cancer immunotherapy strategy using targeted drugs.

  11. Neocortical glial cell numbers in human brains

    DEFF Research Database (Denmark)

    Pelvig, D.P.; Pakkenberg, H.; Stark, A.K.

    2008-01-01

    and neurons and counting were done in each of the four lobes. The study showed that the different subpopulations of glial cells behave differently as a function of age; the number of oligodendrocytes showed a significant 27% decrease over adult life and a strong correlation to the total number of neurons...... while the total astrocyte number is constant through life; finally males have a 28% higher number of neocortical glial cells and a 19% higher neocortical neuron number than females. The overall total number of neocortical neurons and glial cells was 49.3 billion in females and 65.2 billion in males......, a difference of 24% with a high biological variance. These numbers can serve as reference values in quantitative studies of the human neocortex. (C) 2007 Elsevier Inc. All rights reserved Udgivelsesdato: 2008/11...

  12. Characterizing motility dynamics in human RPE cells

    Science.gov (United States)

    Liu, Zhuolin; Kurokawa, Kazuhiro; Zhang, Furu; Miller, Donald T.

    2017-02-01

    Retinal pigment epithelium (RPE) cells are vital to health of the outer retina, however, are often compromised in ageing and ocular diseases that lead to blindness. Early manifestation of RPE disruption occurs at the cellular level, but while in vivo biomarkers at this scale hold considerable promise, RPE cells have proven extremely challenging to image in the living human eye. Recently we addressed this problem by using organelle motility as a novel contrast agent to enhance the RPE cell in conjunction with 3D resolution of adaptive optics-optical coherence tomography (AO-OCT) to section the RPE layer. In this study, we expand on the central novelty of our method - organelle motility - by characterizing the dynamics of the motility in individual RPE cells, important because of its direct link to RPE physiology. To do this, AO-OCT videos of the same retinal patch were acquired at approximately 1 min intervals or less, time stamped, and registered in 3D with sub-cellular accuracy. Motility was quantified by an exponential decay time constant, the time for motility to decorrelate the speckle field across an RPE cell. In two normal subjects, we found the decay time constant to be just 3 seconds, thus indicating rapid motility in normal RPE cells.

  13. Human cell culture in a space bioreactor

    Science.gov (United States)

    Morrison, Dennis R.

    1988-01-01

    Microgravity offers new ways of handling fluids, gases, and growing mammalian cells in efficient suspension cultures. In 1976 bioreactor engineers designed a system using a cylindrical reactor vessel in which the cells and medium are slowly mixed. The reaction chamber is interchangeable and can be used for several types of cell cultures. NASA has methodically developed unique suspension type cell and recovery apparatus culture systems for bioprocess technology experiments and production of biological products in microgravity. The first Space Bioreactor was designed for microprocessor control, no gaseous headspace, circulation and resupply of culture medium, and slow mixing in very low shear regimes. Various ground based bioreactors are being used to test reactor vessel design, on-line sensors, effects of shear, nutrient supply, and waste removal from continuous culture of human cells attached to microcarriers. The small Bioreactor is being constructed for flight experiments in the Shuttle Middeck to verify systems operation under microgravity conditions and to measure the efficiencies of mass transport, gas transfer, oxygen consumption and control of low shear stress on cells.

  14. Synthesis and characterization of novel 2-amino-chromene-nitriles that target Bcl-2 in acute myeloid leukemia cell lines.

    Directory of Open Access Journals (Sweden)

    Hosadurga K Keerthy

    Full Text Available The anti-apoptotic protein Bcl-2 is a well-known and attractive therapeutic target for cancer. In the present study the solution-phase T3P-DMSO mediated efficient synthesis of 2-amino-chromene-3-carbonitriles from alcohols, malanonitrile and phenols is reported. These novel 2-amino-chromene-3-carbonitriles showed cytotoxicity in human acute myeloid leukemia (AML cell lines. Compound 4 g was found to be the most bioactive, decreasing growth and increasing apoptosis of AML cells. Moreover, compound 4 g (at a concentration of 5 µM increased the G2/M and sub-G1 (apoptosis phases of AML cells. The AML cells treated with compound 4 g exhibited decreased levels of Bcl-2 and increased levels of caspase-9. In silico molecular interaction analysis showed that compound 4 g shared a similar global binding motif with navitoclax (another small molecule that binds Bcl-2, however compound 4 g occupies a smaller volume within the P2 hot spot of Bcl-2. The intermolecular π-stacking interaction, direct electrostatic interactions, and docking energy predicted for 4 g in complex with Bcl-2 suggest a strong affinity of the complex, rendering 4 g as a promising Bcl-2 inhibitor for evaluation as a new anticancer agent.

  15. Synthesis and characterization of novel 2-amino-chromene-nitriles that target Bcl-2 in acute myeloid leukemia cell lines.

    Science.gov (United States)

    Keerthy, Hosadurga K; Garg, Manoj; Mohan, Chakrabhavi D; Madan, Vikas; Kanojia, Deepika; Shobith, Rangappa; Nanjundaswamy, Shivananju; Mason, Daniel J; Bender, Andreas; Basappa; Rangappa, Kanchugarakoppal S; Koeffler, H Phillip

    2014-01-01

    The anti-apoptotic protein Bcl-2 is a well-known and attractive therapeutic target for cancer. In the present study the solution-phase T3P-DMSO mediated efficient synthesis of 2-amino-chromene-3-carbonitriles from alcohols, malanonitrile and phenols is reported. These novel 2-amino-chromene-3-carbonitriles showed cytotoxicity in human acute myeloid leukemia (AML) cell lines. Compound 4 g was found to be the most bioactive, decreasing growth and increasing apoptosis of AML cells. Moreover, compound 4 g (at a concentration of 5 µM) increased the G2/M and sub-G1 (apoptosis) phases of AML cells. The AML cells treated with compound 4 g exhibited decreased levels of Bcl-2 and increased levels of caspase-9. In silico molecular interaction analysis showed that compound 4 g shared a similar global binding motif with navitoclax (another small molecule that binds Bcl-2), however compound 4 g occupies a smaller volume within the P2 hot spot of Bcl-2. The intermolecular π-stacking interaction, direct electrostatic interactions, and docking energy predicted for 4 g in complex with Bcl-2 suggest a strong affinity of the complex, rendering 4 g as a promising Bcl-2 inhibitor for evaluation as a new anticancer agent.

  16. Appearance of Human Plasma Cells Following Differentiation of Human B Cells in NOD/SCID Mouse Spleen

    Directory of Open Access Journals (Sweden)

    Kentaro Kikuchi

    2003-01-01

    Full Text Available Relatively little is known for the differentiation and maturation process of human B cells to plasma cells. This is particularly important in reconstitution work involving transfer of autoantibodies. To address this issue, we transplanted human peripheral blood mononuclear cells (PBMC directly into the spleen of irradiated NOD/SCID mice depleted of natural killer cell activity. Within 6 weeks, naïve B cells differentiated into memory B cells and, importantly, the numbers of human CD138+ plasma cells in spleen increased by 100 fold after transplantation. Plasma cell numbers correlated with the detection of human IgM and IgG in serum, indicating that human B cells had differentiated into mature plasma cells in the murine spleen. In addition to CD19+ plasma cells, a distinct CD19- plasma cell population was detected, suggesting that downregulation of CD19 associated with maturation of plasma cells occurred. When purified human B cells were transplanted, those findings were not observed. Our results indicate that differentiation and maturation of human B cells and plasma cells can be investigated by transplantation of human PBMC into the spleen of NOD/SCID mice. The model will be useful for studying the differentiation of human B cells and generation of plasma cells.

  17. Chromatin organization as a possible factor in the control of susceptibility to radiation-induced AML in mice

    Science.gov (United States)

    Maranon, David G.

    The studies described in this dissertation involve the use and comparison of two mouse strains: one sensitive (CBA/CaJ) and another resistant (C57BL/6J) to radiation-induced acute myeloid leukemia (AML). The purpose of these studies was to identify factors that may account for the large difference in the susceptibility of these strains to radiation-induced AML. The present study was initiated to determine whether the distances between breakpoint clusters on chromosome 2 are in closer proximity in the bone marrow cells of the CBA/CaJ mouse strain than in the C57BL/6J strain. Bacterial artificial chromosomes (BACs) were selected as markers of the central portion of the proximal and distal deletion breakpoint clusters as well as mdr on chromosome 2, where the preponderance of breaks occurs. Distance measurements were made by three dimensional fluorescent in situ hybridization (3DFISH) image analysis of hundreds of cells using Metamorph and ImageJ for data collection and Autoquant software for deconvolution and reconstruction of the three dimensional cell nuclei. Comparing bone marrow cells of CBA/CaJ and C57BL/6J mice, no differences were found between the proximity of the two regions represented for the selected markers compared in both murine strains. For the markers chosen the distribution of the distances showed similarities between the same cell types from both mouse strains; namely, fibroblasts, whole bone marrow (WBM), and hematopoietic stem cells (HSC). However, there was not found a change in the distance distributions toward the closer distances expected between the clusters in HSC and WBM compared with fibroblasts in both mouse strains. There was; however, a tissue-dependent distance distribution between the markers Specifically, the average distances of the clusters in fibroblasts (2.55 um for CBA/CaJ and 3.09 um for C57BL/6) were larger than the distance in blood cells (1.74 um in BM and 1.53 um in HSC for CBA/CaJ; and 1.79 um in BM and 1.77 um in HSC for

  18. Extramedullary Acute Myeloid Leukemia (AML: Leukemic Pleural Effusion, Case Report and Review of the Literature

    Directory of Open Access Journals (Sweden)

    Naveen ePemmaraju

    2014-06-01

    Full Text Available Objective and Importance: Malignant pleural effusions occur in the setting of both solid and hematologic malignancies. Pleural effusion caused by leukemic infiltration is an unusual extramedullary manifestation of acute myeloid leukemia (AML with fewer than 20 cases reported.1-11 We report a case of pericardial and pleural effusions in a patient with AML and review the literature. Clinical presentation: In this case, a 55 year old man with previous history of myeloproliferative neoplasm (MPN experienced transformation AML, heralded by appearance of leukemic pleural effusions. The patient was identified to have leukemic pleural effusion based upon extended cytogenetic analysis of the pleural fluid, as morphologic analysis alone was insufficient. Intervention: The patient was treated with hypomethylator-based and intensive chemotherapy strategies, both of which maintained resolution of the effusions in the remission setting. Conclusion: Due to the rarity of diagnosis of leukemic pleural effusions, both cytogenetic and fluorescence in situ hybridization (FISH testing are recommended. Futhermore, systemic chemotherapy directed at the AML can lead to complete resolution of leukemic pleural effusions. Objective and ImportancePleural effusion caused by leukemic infiltration is an unusual extramedullary manifestation of acute myeloid leukemia (AML, but may be more common than previously thought. Fewer than 20 cases have been reported.1-11 We report a case of pericardial and pleural effusions in a patient with AML and review the literature.

  19. Human somatic cell nuclear transfer is alive and well.

    Science.gov (United States)

    Cibelli, Jose B

    2014-06-05

    In this issue, Chung et al. (2014) generate human embryonic stem cells by fusing an adult somatic cell to a previously enucleated human oocyte, in agreement with recent reports by the Mitalipov and Egli groups. We can now safely say that human somatic cell nuclear transfer is alive and well.

  20. Inhibition of DNA and Histone Methylation by 5-Aza-2′-Deoxycytidine (Decitabine) and 3-Deazaneplanocin-A on Antineoplastic Action and Gene Expression in Myeloid Leukemic Cells

    Science.gov (United States)

    Momparler, Richard L.; Côté, Sylvie; Momparler, Louise F.; Idaghdour, Youssef

    2017-01-01

    Epigenetic alterations play an important role in the development of acute myeloid leukemia (AML) by silencing of genes that suppress leukemogenesis and differentiation. One of the key epigenetic changes in AML is gene silencing by DNA methylation. The importance of this alteration is illustrated by the induction of remissions in AML by 5-aza-2′-deoxycytidine (5-AZA-CdR, decitabine), a potent inhibitor of DNA methylation. However, most patients induced into remission by 5-AZA-CdR will relapse, suggesting that a second agent should be sought to increase the efficacy of this epigenetic therapy. An interesting candidate for this purpose is 3-deazaneplanocin A (DZNep). This analog inhibits EZH2, a histone methyltransferase that trimethylates lysine 27 histone H3 (H3K27me3), a marker for gene silencing. This second epigenetic silencing mechanism also plays an important role in leukemogenesis as shown in preclinical studies where DZNep exhibits potent inhibition of colony formation by AML cells. We reported previously that 5-AZA-CdR in combination with DZNep exhibits a synergistic antineoplastic action against human HL-60 AML cells and the synergistic activation of several tumor suppressor genes. In this report, we showed that this combination also induced a synergistic activation of apoptosis in HL-60 cells. The synergistic antineoplastic action of 5-AZA-CdR plus DZNep was also observed on a second human myeloid leukemia cell line, AML-3. In addition, 5-AZA-CdR in combination with the specific inhibitors of EZH2, GSK-126, or GSK-343, also exhibited a synergistic antineoplastic action on both HL-60 and AML-3. The combined action of 5-AZA-CdR and DZNep on global gene expression in HL-60 cells was investigated in greater depth using RNA sequencing analysis. We observed that this combination of epigenetic agents exhibited a synergistic activation of hundreds of genes. The synergistic activation of so many genes that suppress malignancy by 5-AZA-CdR plus DZNep suggests that

  1. The association between human papillomavirus and oropharyngeal squamous cell Carcinoma

    DEFF Research Database (Denmark)

    Walvik, Lena; Svensson, Amanda Björk; Friborg, Jeppe

    2016-01-01

    There is emerging evidence of the association between human papillomavirus and a subset of head and neck cancers. However, the role of human papillomavirus as a causal factor is still debated. This review addresses the association between human papillomavirus and oropharyngeal squamous cell...... of well-defined premalignant lesions. However, a causal relationship between human papillomavirus infection and oropharyngeal squamous cell carcinoma seems evident....

  2. Cell cycle regulation in human embryonic stem cells: links to adaptation to cell culture.

    Science.gov (United States)

    Barta, Tomas; Dolezalova, Dasa; Holubcova, Zuzana; Hampl, Ales

    2013-03-01

    Cell cycle represents not only a tightly orchestrated mechanism of cell replication and cell division but it also plays an important role in regulation of cell fate decision. Particularly in the context of pluripotent stem cells or multipotent progenitor cells, regulation of cell fate decision is of paramount importance. It has been shown that human embryonic stem cells (hESCs) show unique cell cycle characteristics, such as short doubling time due to abbreviated G1 phase; these properties change with the onset of differentiation. This review summarizes the current understanding of cell cycle regulation in hESCs. We discuss cell cycle properties as well as regulatory machinery governing cell cycle progression of undifferentiated hESCs. Additionally, we provide evidence that long-term culture of hESCs is accompanied by changes in cell cycle properties as well as configuration of several cell cycle regulatory molecules.

  3. A New Case of dic(1;15(p11;p11 in AML M1: Apropos of a Case and a Review of the Literature

    Directory of Open Access Journals (Sweden)

    Deniz Gören Şahin

    2013-01-01

    Full Text Available Acute myelogenous leukemia (AML develops as the consequence of a series of genetic changes in a hematopoietic precursor cell. Specific cytogenetic abnormalities have been identified by karyotype analysis in AML. One of the rare chromosomal abnormalities is a dicentric chromosome, which is defined as an aberrant chromosome having two centromeres. In the literature, a limited number of cases have been reported with dic(1;15 in myeloid disorders, but only one case has been reported with in acute megakaryoblastic leukemia. Herein, we report a case of acute myelogenous leukemia without maturation with a dic(1;15(p11;p11, resulting in trisomy of the long arm of chromosome 1. To date, this is the second case of dic(1;15 in acute myelogenous leukemia and the first case in acute myeloblastic leukemia without maturation.

  4. Glycomics of human embryonic stem cells and human induced pluripotent stem cells.

    Science.gov (United States)

    Furukawa, Jun-Ichi; Okada, Kazue; Shinohara, Yasuro

    2016-10-01

    Most cells are coated by a dense glycocalyx composed of glycoconjugates such as glycosphingolipids, glycoproteins, and proteoglycans. The overall glycomic profile is believed to be crucial for the diverse roles of glycans, which are mediated by specific interactions that regulate cell-cell adhesion, the immune response, microbial pathogenesis, and other cellular events. Many cell surface markers were discovered and identified as glycoconjugates such as stage-specific embryonic antigen, Tra-1-60/81 and various other cell surface molecules (e.g., cluster of differentiation). Recent progress in the development of analytical methodologies and strategies has begun to clarify the cellular glycomics of various cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). The glycomic profiles of these cells are highly cell type-specific and reflect cellular alterations, such as development, differentiation and cancerous change. In this mini review, we briefly summarize the glycosylation spectra specific to hESCs and hiPSCs, which cover glycans of all major glycoconjugates (i.e., glycosphingolipids, N- and O-glycans of glycoproteins, and glycosaminoglycans) and free oligosaccharides.

  5. Isolation and characterization of human spermatogonial stem cells

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    Liu Shixue

    2011-10-01

    Full Text Available Abstract Background To isolate and characterization of human spermatogonial stem cells from stem spermatogonium. Methods The disassociation of spermatogonial stem cells (SSCs were performed using enzymatic digestion of type I collagenase and trypsin. The SSCs were isolated by using Percoll density gradient centrifugation, followed by differential surface-attachment method. Octamer-4(OCT4-positive SSC cells were further identified using immunofluorescence staining and flow cytometry technques. The purity of the human SSCs was also determined, and a co-culture system for SSCs and Sertoli cells was established. Results The cell viability was 91.07% for the suspension of human spermatogonial stem cells dissociated using a two-step enzymatic digestion process. The cells isolated from Percoll density gradient coupled with differential surface-attachement purification were OCT4 positive, indicating the cells were human spermatogonial stem cells. The purity of isolated human spermatogonial stem cells was 86.7% as assessed by flow cytometry. The isolated SSCs were shown to form stable human spermatogonial stem cell colonies on the feeder layer of the Sertoli cells. Conclusions The two-step enzyme digestion (by type I collagenase and trypsin process is an economical, simple and reproducible technique for isolating human spermatogonial stem cells. With little contamination and less cell damage, this method facilitates isolated human spermatogonial stem cells to form a stable cell colony on the supporting cell layer.

  6. Cell entry by human pathogenic arenaviruses.

    Science.gov (United States)

    Rojek, Jillian M; Kunz, Stefan

    2008-04-01

    The arenaviruses Lassa virus (LASV) in Africa and Machupo (MACV), Guanarito (GTOV) and Junin viruses (JUNV) in South America cause severe haemorrhagic fevers in humans with fatality rates of 15-35%. The present review focuses on the first steps of infection with human pathogenic arenaviruses, the interaction with their cellular receptor molecules and subsequent entry into the host cell. While similarities exist in genomic organization, structure and clinical disease caused by pathogenic Old World and New World arenaviruses these pathogens use different primary receptors. The Old World arenaviruses employ alpha-dystroglycan, a cellular receptor for proteins of the extracellular matrix, and the human pathogenic New World arenaviruses use the cellular cargo receptor transferrin receptor 1. While the New World arenavirus JUNV enters cells via clathrin-dependent endocytosis, evidence occurred for clathrin-independent entry of the prototypic Old World arenavirus lymphocytic choriomeningitis virus. Upon internalization, arenaviruses are delivered to the endosome, where pH-dependent membrane fusion is mediated by the envelope glycoprotein (GP). While arenavirus GPs share characteristics with class I fusion GPs of other enveloped viruses, unusual mechanistic features of GP-mediated membrane fusion have recently been discovered for arenaviruses with important implications for viral entry.

  7. Markers of T Cell Senescence in Humans

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    Weili Xu

    2017-08-01

    Full Text Available Many countries are facing the aging of their population, and many more will face a similar obstacle in the near future, which could be a burden to many healthcare systems. Increased susceptibility to infections, cardiovascular and neurodegenerative disease, cancer as well as reduced efficacy of vaccination are important matters for researchers in the field of aging. As older adults show higher prevalence for a variety of diseases, this also implies higher risk of complications, including nosocomial infections, slower recovery and sequels that may reduce the autonomy and overall quality of life of older adults. The age-related effects on the immune system termed as “immunosenescence” can be exemplified by the reported hypo-responsiveness to influenza vaccination of the elderly. T cells, which belong to the adaptive arm of the immune system, have been extensively studied and the knowledge gathered enables a better understanding of how the immune system may be affected after acute/chronic infections and how this matters in the long run. In this review, we will focus on T cells and discuss the surface and molecular markers that are associated with T cell senescence. We will also look at the implications that senescent T cells could have on human health and diseases. Finally, we will discuss the benefits of having these markers for investigators and the future work that is needed to advance the field of T cell senescence markers.

  8. Human embryonic stem cells and microenvironment

    Directory of Open Access Journals (Sweden)

    Banu İskender

    2014-09-01

    Full Text Available Human embryonic stem cells (hESCs possess a great potential in the field of regenerative medicine by their virtue of pluripotent potential with indefinite proliferation capabilities. They can self renew themselves and differentiate into three embryonic germ layers. Although they are conventionally grown on mitotically inactivated mouse feeder cells, there are in vitro culture systems utilizing feeder cells of human origin in order to prevent cross-species contamination. Recently established in vitro culture systems suggested that direct interaction with feeder cells is not necessary but rather attachment to a substrate is required to ensure long-term, efficient hESC culture in vitro. This substrate is usually composed of a mixture of extracellular matrix components representing in vivo natural niche. In hESC biology, the mechanism of interaction of hESCs with extracellular matrix molecules remained insufficiently explored area of research due to their transient nature of interaction with the in vivo niche. However, an in vitro culture system established using extracellular matrix molecules may provide a safer alternative to culture systems with feeder cells while paving the way to Good Manufacturing Practice-GMP production of hESCs for therapeutic purposes. Therefore, it is essential to study the interaction of extracellular matrix molecules with hESCs in order to standardize in vitro culture systems for large-scale production of hESCs in a less labor-intensive way. This would not only provide valuable information regarding the mechanisms that control pluripotency but also serve to dissect the molecular signaling pathways of directed differentiation for prospective therapeutic applications in the future. J Clin Exp Invest 2014; 5 (3: 486-495

  9. Arecoline is cytotoxic for human endothelial cells.

    Science.gov (United States)

    Ullah, Mafaz; Cox, Stephen; Kelly, Elizabeth; Boadle, Ross; Zoellner, Hans

    2014-11-01

    Oral submucous fibrosis is a pre-malignant fibrotic condition caused by areca nut use and involves reduced mucosal vascularity. Arecoline is the principal areca nut alkaloid and is cytotoxic for epithelium and fibroblasts. Endothelial cell cycle arrest is reported on exposure to arecoline, as is cytotoxicity for endothelial-lung carcinoma hybrid cells. We here describe cytotoxicity for primary human endothelial cultures from seven separate donors. Human umbilical vein endothelial cells were exposed to increasing concentrations of arecoline and examined by: phase-contrast microscopy, haemocytometer counts, transmission electron microscopy, lactate dehydrogenase release and the methyl-thiazol-tetrazolium assay. Vacuolation and detachment of endothelium were observed at and above arecoline concentrations of 333 μg/ml or more. Ultrastructural features of cellular stress were seen after 24-h treatment with 111 μg/ml arecoline and included reduced ribosomal studding of endoplasmic reticulum, increased autophagolysosomal structures, increased vacuolation and reduced mitochondrial cristae with slight swelling. Similar changes were seen at 4 h with arecoline at 333 μg/ml or above, but with more severe mitochondrial changes including increased electron density of mitochondrial matrix and greater cristal swelling, while by 24 h, these cells were frankly necrotic. Haemocytometer counts were paralleled by both lactate dehydrogenase release and the methyl-thiazol-tetrazolium assays. Arecoline is cytotoxic via necrosis for endothelium, while biochemical assays indicate no appreciable cellular leakage before death and detachment, as well as no clear effect on mitochondrial function in viable cells. Arecoline toxicity may thus contribute to reduced vascularity in oral submucous fibrosis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. Lysosomal disruption preferentially targets acute myeloid leukemia cells and progenitors

    Science.gov (United States)

    Sukhai, Mahadeo A.; Prabha, Swayam; Hurren, Rose; Rutledge, Angela C.; Lee, Anna Y.; Sriskanthadevan, Shrivani; Sun, Hong; Wang, Xiaoming; Skrtic, Marko; Seneviratne, Ayesh; Cusimano, Maria; Jhas, Bozhena; Gronda, Marcela; MacLean, Neil; Cho, Eunice E.; Spagnuolo, Paul A.; Sharmeen, Sumaiya; Gebbia, Marinella; Urbanus, Malene; Eppert, Kolja; Dissanayake, Dilan; Jonet, Alexia; Dassonville-Klimpt, Alexandra; Li, Xiaoming; Datti, Alessandro; Ohashi, Pamela S.; Wrana, Jeff; Rogers, Ian; Sonnet, Pascal; Ellis, William Y.; Corey, Seth J.; Eaves, Connie; Minden, Mark D.; Wang, Jean C.Y.; Dick, John E.; Nislow, Corey; Giaever, Guri; Schimmer, Aaron D.

    2012-01-01

    Despite efforts to understand and treat acute myeloid leukemia (AML), there remains a need for more comprehensive therapies to prevent AML-associated relapses. To identify new therapeutic strategies for AML, we screened a library of on- and off-patent drugs and identified the antimalarial agent mefloquine as a compound that selectively kills AML cells and AML stem cells in a panel of leukemia cell lines and in mice. Using a yeast genome-wide functional screen for mefloquine sensitizers, we identified genes associated with the yeast vacuole, the homolog of the mammalian lysosome. Consistent with this, we determined that mefloquine disrupts lysosomes, directly permeabilizes the lysosome membrane, and releases cathepsins into the cytosol. Knockdown of the lysosomal membrane proteins LAMP1 and LAMP2 resulted in decreased cell viability, as did treatment of AML cells with known lysosome disrupters. Highlighting a potential therapeutic rationale for this strategy, leukemic cells had significantly larger lysosomes compared with normal cells, and leukemia-initiating cells overexpressed lysosomal biogenesis genes. These results demonstrate that lysosomal disruption preferentially targets AML cells and AML progenitor cells, providing a rationale for testing lysosomal disruption as a novel therapeutic strategy for AML. PMID:23202731

  11. Nuclear factor E2-related factor 2 knockdown enhances glucose uptake and alters glucose metabolism in AML12 hepatocytes.

    Science.gov (United States)

    Yuan, Xiaoyang; Huang, Huijing; Huang, Yi; Wang, Jinli; Yan, Jinhua; Ding, Ling; Zhang, Cuntai; Zhang, Le

    2017-05-01

    Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor known to induce the expression of a variety of antioxidant and detoxification genes. Recently, increasing evidence has revealed roles for Nrf2 in glucose, lipid, and energy metabolism; however, the exact functions of Nrf2 in hepatocyte biology are largely unclear. In the current study, the transient knockdown of Nrf2 via siRNA transfection enhanced the glucose uptake of fasting AML12 hepatocytes to 325.3 ± 11.1% ( P glucose metabolism were then examined in AML12 cells under both high-glucose (33 mmol/L) and low-glucose (4.5 mmol/L) conditions. NK lowered the gene and protein expression of the anti-oxidases heme oxygenase-1 and NAD(P)H: quinone oxidoreductase 1 and increased p-eukaryotic initiation factor-2α(S51), p-nuclear factor-κB p65(S276), and its downstream proinflammatory factors, including interleukin-1 beta, tumor necrosis factor-α, matrix metalloproteinase 2, and matrix metalloproteinase 9, at the protein level. NK also altered the protein expression of fibroblast growth factor 21, glucose transporter type 4, insulin-like growth factor 1, forkhead box protein O1, p-AKT(S473), and p-GSK3α/β(Y279/Y216), which are involved in glucose uptake, glycogenesis, and gluconeogenesis in AML12 cells. Our results provide a comprehensive understanding of the central role of Nrf2 in the regulation of glucose metabolism in AML12 hepatocytes, in addition to its classical roles in the regulation of redox signaling, endoplasmic reticulum stress and proinflammatory responses, and support the potential of Nrf2 as a therapeutic target for the prevention and treatment of obesity and other associated metabolic syndromes. Impact statement Increasing evidence supports the complexity of Nrf2 functions beyond the antioxidant and detoxification response. Previous in vivo studies employing either Nrf2-knockout or Nrf2-activated mice have achieved a similar endpoint: protection against an obese and

  12. Identification of a candidate stem cell in human gallbladder

    Directory of Open Access Journals (Sweden)

    Rohan Manohar

    2015-05-01

    In conclusion, we have isolated a distinct clonogenic population of epithelial cells from primary human fetal gallbladder with stem cell characteristics and found it to be unique compared to IHBD cells.

  13. EVI1 inhibits apoptosis induced by antileukemic drugs via upregulation of CDKN1A/p21/WAF in human myeloid cells.

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    Anna Rommer

    Full Text Available Overexpression of ecotropic viral integration site 1 (EVI1 is associated with aggressive disease in acute myeloid leukemia (AML. Despite of its clinical importance, little is known about the mechanism through which EVI1 confers resistance to antileukemic drugs. Here, we show that a human myeloid cell line constitutively overexpressing EVI1 after infection with a retroviral vector (U937_EVI1 was partially resistant to etoposide and daunorubicin as compared to empty vector infected control cells (U937_vec. Similarly, inducible expression of EVI1 in HL-60 cells decreased their sensitivity to daunorubicin. Gene expression microarray analyses of U937_EVI1 and U937_vec cells cultured in the absence or presence of etoposide showed that 77 and 419 genes were regulated by EVI1 and etoposide, respectively. Notably, mRNA levels of 26 of these genes were altered by both stimuli, indicating that EVI1 regulated genes were strongly enriched among etoposide regulated genes and vice versa. One of the genes that were induced by both EVI1 and etoposide was CDKN1A/p21/WAF, which in addition to its function as a cell cycle regulator plays an important role in conferring chemotherapy resistance in various tumor types. Indeed, overexpression of CDKN1A in U937 cells mimicked the phenotype of EVI1 overexpression, similarly conferring partial resistance to antileukemic drugs.

  14. Antiproliferative Effects of Drugs on Endothelial and Osteoblastic Cells and Altered Release of Angioregulatory Mediators by Endothelial Cells

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    Hilde Kvestad

    2014-01-01

    Full Text Available The combined use of the histone deacetylase inhibitor valproic acid (VPA, the retinoic acid receptor-α agonist all-trans retinoic acid (ATRA, and the deoxyribonucleic acid polymerase-α inhibitor cytarabine (Ara-C is now considered for disease-stabilizing treatment of acute myeloid leukemia (AML. Leukemogenesis and leukemia cell chemoresistance seem to be supported by neighbouring stromal cells in the bone marrow, and we have therefore investigated the effects of these drugs on primary human endothelial cells and the osteoblastic Cal72 cell line. The results show that VPA and Ara-C have antiproliferative effects, and the antiproliferative/cytotoxic effect of Ara-C was seen at low concentrations corresponding to serum levels found during low-dose in vivo treatment. Furthermore, in functional assays of endothelial migration and tube formation VPA elicited an antiangiogenic effect, whereas ATRA elicited a proangiogenic effect. Finally, VPA and ATRA altered the endothelial cell release of angiogenic mediators; ATRA increased levels of CXCL8, PDGF-AA, and VEGF-D, while VPA decreased VEGF-D and PDGF-AA/BB levels and both drugs reduced MMP-2 levels. Several of these mediators can enhance AML cell proliferation and/or are involved in AML-induced bone marrow angiogenesis, and direct pharmacological effects on stromal cells may thus indirectly contribute to the overall antileukemic activity of this triple drug combination.

  15. Cell cycle regulation by glucosamine in human pulmonary epithelial cells.

    Science.gov (United States)

    Chuang, Kun-Han; Lu, Chih-Shen; Kou, Yu Ru; Wu, Yuh-Lin

    2013-04-01

    Airway epithelial cells play an important role against intruding pathogens. Glucosamine, a commonly used supplemental compound, has recently begun to be regarded as a potential anti-inflammatory molecule. This study aimed to uncover how glucosamine impacts on cellular proliferation in human alveolar epithelial cells (A549) and bronchial epithelial cells (HBECs). With trypan blue-exclusion assay, we observed that glucosamine (10, 20, 50 mM) caused a decrease in cell number at 24 and 48 h; with a flow cytometric analysis, we also noted an enhanced cell accumulation within the G(0)/G(1) phase at 24 h and induction of late apoptosis at 24 and 48 h by glucosamine (10, 20, 50 mM) in A549 cells and HBECs. Examination of phosphorylation in retinoblastoma (Rb) protein, we found an inhibitory effect by glucosamine at 20 and 50 mM. Glucosamine at 50 mM was demonstrated to elevate both the mRNA and protein expression of p53 and heme oxygenase-1 (HO-1), but also caused a reduction in p21 protein expression. In addition, glucosamine attenuated p21 protein stability via the proteasomal proteolytic pathway, as well as inducing p21 nuclear accumulation. Altogether, our results suggest that a high dose of glucosamine may inhibit cell proliferation through apoptosis and disturb cell cycle progression with a halt at G(0)/G(1) phase, and that this occurs, at least in part, by a reduction in Rb phosphorylation together with modulation of p21, p53 and HO-1 expression, and nuclear p21 accumulation.

  16. Clinical and biological impact of TET2 mutations and expression in younger adult AML patients treated within the EORTC/GIMEMA AML-12 clinical trial.

    Science.gov (United States)

    Aslanyan, Mariam G; Kroeze, Leonie I; Langemeijer, Saskia M C; Koorenhof-Scheele, Theresia N; Massop, Marion; van Hoogen, Patricia; Stevens-Linders, Ellen; van de Locht, Louis T; Tönnissen, Evelyn; van der Heijden, Adrian; da Silva-Coelho, Pedro; Cilloni, Daniela; Saglio, Giuseppe; Marie, Jean-Pierre; Tang, Ruoping; Labar, Boris; Amadori, Sergio; Muus, Petra; Willemze, Roel; Marijt, Erik W A; de Witte, Theo; van der Reijden, Bert A; Suciu, Stefan; Jansen, Joop H

    2014-08-01

    We assessed the prognostic impact of TET2 mutations and mRNA expression in a prospective cohort of 357 adult AML patients Adulto (GIMEMA) AML-12 06991 clinical trial. In addition the co-occurrence with other genetic defects and the functional consequences of TET2 mutations were investigated. TET2 mutations occurred in 7.6 % of the patients and were an independent marker of poor prognosis (p = 0.024). TET2 and IDH1/2 mutations strongly associated with aberrations in the DNA methyltransferase DNMT3A. Functional studies confirmed previous work that neither nonsense truncations, nor missense TET2 mutations, induced 5-hydroxymethylcytosine formation. In addition, we now show that mutant TET2 forms did not act in a dominant negative manner when co-expressed with the wild-type protein. Finally, as loss-of-function TET2 mutations predicted poor outcome, we questioned whether low TET2 mRNA expression in cases of AML without TET2 mutations would affect overall survival. Notably, also AML patients with low TET2 mRNA expression levels showed inferior overall survival.

  17. Treatment for myeloid leukaemia of Down syndrome: population-based experience in the UK and results from the Medical Research Council AML 10 and AML 12 trials.

    NARCIS (Netherlands)

    Rao, A.; Hills, R.K.; Stiller, C.; Gibson, B.E.; Graaf, S.S.N. de; Hann, I.M.; O'Marcaigh, A.; Wheatley, K.; Webb, D.K.

    2006-01-01

    Down syndrome (DS) children are at an increased risk of developing myelodysplasia and acute myeloid leukaemia (AML). We retrospectively analysed the population-based data on 81 children with myeloid leukaemia of Down syndrome (ML-DS) from the UK National Registry of Childhood Tumours and experience

  18. Human embryonic stem cells and patent protection

    Directory of Open Access Journals (Sweden)

    Radovanović Sanja M.

    2015-01-01

    Full Text Available Given the importance of biotechnological research in modern diagnostics and therapeutics, on the one hand, and stimulative function of a patent, on the other hand, this work deals with the question of the possibility of pa-tent protection of human embryonic stem cells. Taking into account that this is a biotechnological invention, the key question that this paper highlights is the interpretation of the provisions of their patentability. Namely, thanks to the advanced methods of isolation, purification and preparation for implementation, modern patent systems do not exclude a priori living organisms from patent protection. Therefore, the analysis of representative administrative decisions or court rulings sought to define the criteria that would be applied in order to give patent protection to a certain biotechnological invention (stem cells while others do not.

  19. Allium compounds, dipropyl and dimethyl thiosulfinates as antiproliferative and differentiating agents of human acute myeloid leukemia cell lines

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    Faten Merhi

    2008-08-01

    Full Text Available Faten Merhi1, Jacques Auger2, Francine Rendu1, Brigitte Bauvois11UMR 7131 UPMC Paris Universitas/CNRS, Groupe Hospitalier Broussais-HEGP, Paris, France; 2University F. Rabelais, IRBI, UPRESA CNRS 6035, Tours, FranceAbstract: Epidemiologic studies support the premise that Allium vegetables may lower the risk of cancers. The beneficial effects appear related to the organosulfur products generated upon processing of Allium. Leukemia cells from patients with acute myeloid leukemia (AML display high proliferative capacity and have a reduced capacity of undergoing apoptosis and maturation. Whether the sulfur-containing molecules thiosulfinates (TS, diallyl TS (All2TS, dipropyl TS (Pr2TS and dimethyl TS (Me2TS, are able to exert chemopreventative activity against AML is presently unknown. The present study was an evaluation of proliferation, cytotoxicity, differentiation and secretion of AML cell lines (U937, NB4, HL-60, MonoMac-6 in response to treatment with these TS and their related sulfides (diallylsulfide, diallyl disulfide, dipropyl disulfide, dimethyl disulfide. As assessed by flow cytometry, ELISA, gelatin zymogaphy and RT-PCR, we showed that Pr2TS and Me2TS, but not All2TS and sulfides, 1 inhibited cell proliferation in dose- and time-dependent manner and this process was neither due to cytotoxicity nor apoptosis, 2 induced macrophage maturation, and 3 inhibited the levels of secreted MMP-9 (protein and activity and TNF-α protein, without altering mRNA levels. By establishing for the first time that Pr2TS and Me2TS affect proliferation, differentiation and secretion of leukemic cell lines, this study provides the opportunity to explore the potential efficiency of these molecules in AML.Keywords: acute myeloid leukemia, thiosulfinate, proliferation, differentiation, matrix metalloproteinase-9

  20. Generation of human induced pluripotent stem cells from dermal fibroblasts

    OpenAIRE

    2008-01-01

    The generation of patient-specific pluripotent stem cells has the potential to accelerate the implementation of stem cells for clinical treatment of degenerative diseases. Technologies including somatic cell nuclear transfer and cell fusion might generate such cells but are hindered by issues that might prevent them from being used clinically. Here, we describe methods to use dermal fibroblasts easily obtained from an individual human to generate human induced pluripotent stem (iPS) cells by ...

  1. Restriction of human adenovirus replication in Chinese hamster cell lines and their hybrids with human cells.

    Science.gov (United States)

    Radna, R L; Foellmer, B; Feldman, L A; Francke, U; Ozer, H L

    1987-11-01

    We have found that the replication of human adenovirus (Ad2) is restricted in multiple Chinese hamster cell lines including CHO and V79. The major site of restriction involves differential accumulation of late viral proteins as demonstrated by immunofluorescence assay and polyacrylamide gel electrophoresis with and without prior immunoprecipitation. Synthesis of fiber and penton base are markedly reduced, whereas others, such as the 100K polypeptide, are synthesized efficiently. This pattern of restriction is similar to that previously reported for Ad2 infection of several monkey cell lines; however, the restriction is more marked in the Chinese hamster cell lines. The restriction is most likely due to a deficient cellular function since stable cell hybrids between V79 or CHO and human cells are permissive for virus replication. By analysis of a series of hybrids with reduced numbers of human chromosomes, fiber synthesis was correlated with the presence of the short arm of human chromosome 3. More hybrids showed restoration of fiber synthesis than production of progeny virus, suggesting that more than one unlinked function is required for the latter.

  2. Phenotypic changes of human cells in human-rat liver during partial hepatectomy-induced regeneration

    Institute of Scientific and Technical Information of China (English)

    Yan Sun; Dong Xiao; Hong-An Li; Jin-Fang Jiang; Qing Li; Ruo-Shuang Zhang; Xi-Gu Chen

    2009-01-01

    AIM: To examine the human hepatic parenchymal and stromal components in rat liver and the phenotypic changes of human cells in liver of human-rat chimera (HRC) generated by in utero transplantation of human cells during partial hepatectomy (PHx)-induced liver regeneration. METHODS: Human hepatic parenchymal and stromal components and phenotypic changes of human cells during liver regeneration were examined by flow cytometry, in situ hybridization and immunohistochemistry. RESULTS: ISH analysis demonstrated human Alupositive cells in hepatic parenchyma and stroma of recipient liver. Functional human hepatocytes generated in this model potentially constituted human hepatic functional units with the presence of donor-derived human endothelial and biliary duct cells in host liver. Alpha fetoprotein (AFP)+, CD34+ and CD45+ cells were observed in the chimeric liver on day 10 after PHxinduced liver regeneration and then disappeared in PHx group, but not in non-PHx group, suggesting that dynamic phenotypic changes of human cells expressing AFP, CD34 and CD45 cells may occur during the chimeric liver regeneration. Additionally, immunostaining for human proliferating cell nuclear antigen (PCNA) showed that the number of PCNA-positive cells in the chimeric liver of PHx group was markedly increased, as compared to that of control group, indicating that donor-derived human cells are actively proliferated during PHx-induced regeneration of HRC liver.

  3. HIF induces human embryonic stem cell markers in cancer cells.

    Science.gov (United States)

    Mathieu, Julie; Zhang, Zhan; Zhou, Wenyu; Wang, Amy J; Heddleston, John M; Pinna, Claudia M A; Hubaud, Alexis; Stadler, Bradford; Choi, Michael; Bar, Merav; Tewari, Muneesh; Liu, Alvin; Vessella, Robert; Rostomily, Robert; Born, Donald; Horwitz, Marshall; Ware, Carol; Blau, C Anthony; Cleary, Michele A; Rich, Jeremy N; Ruohola-Baker, Hannele

    2011-07-01

    Low oxygen levels have been shown to promote self-renewal in many stem cells. In tumors, hypoxia is associated with aggressive disease course and poor clinical outcomes. Furthermore, many aggressive tumors have been shown to display gene expression signatures characteristic of human embryonic stem cells (hESC). We now tested whether hypoxia might be responsible for the hESC signature observed in aggressive tumors. We show that hypoxia, through hypoxia-inducible factor (HIF), can induce an hESC-like transcriptional program, including the induced pluripotent stem cell (iPSC) inducers, OCT4, NANOG, SOX2, KLF4, cMYC, and microRNA-302 in 11 cancer cell lines (from prostate, brain, kidney, cervix, lung, colon, liver, and breast tumors). Furthermore, nondegradable forms of HIFα, combined with the traditional iPSC inducers, are highly efficient in generating A549 iPSC-like colonies that have high tumorigenic capacity. To test potential correlation between iPSC inducers and HIF expression in primary tumors, we analyzed primary prostate tumors and found a significant correlation between NANOG-, OCT4-, and HIF1α-positive regions. Furthermore, NANOG and OCT4 expressions positively correlated with increased prostate tumor Gleason score. In primary glioma-derived CD133 negative cells, hypoxia was able to induce neurospheres and hESC markers. Together, these findings suggest that HIF targets may act as key inducers of a dynamic state of stemness in pathologic conditions.

  4. Influence of BCL2-938C>A and BAX-248G>A promoter polymorphisms in the development of AML: case-control study from South India.

    Science.gov (United States)

    Cingeetham, Anuradha; Vuree, Sugunakar; Dunna, Nageswara Rao; Gorre, Manjula; Nanchari, Santhoshi Rani; Edathara, Prajitha Mohandas; Meka, Phannibhushann; Annamaneni, Sandhya; Digumarthi, Raghunadharao; Sinha, Sudha; Satti, Vishnupriya

    2015-09-01

    B-cell lymphoma 2 (BCL2) and BCL2-associated X protein (BAX) proteins are anti-apoptotic and pro-apoptotic determinants of mitochondrial-mediated apoptosis, and their relative expression determines the cell fate. The promoter polymorphisms in these genes were shown to alter the protein function or expression and exert an impact on apoptosis regulation. Deregulation in the expression of any of these genes leads to disruption of cellular homeostasis and malignant transformation. The present study was aimed to determine the association of BCL2-938C>A and BAX-248G>A promoter polymorphisms with origin and progression of acute myeloid leukemia (AML). We also have performed combined genotype analysis to evaluate the cumulative effect of risk genotypes in the AML development. These polymorphisms were genotyped by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) in 221 AML patients and 305 age- and sex-matched healthy controls. Our study revealed that BCL2-938CA (p = 0.018) and BAX-248GG (0.043) genotypes were significantly associated with increased risk for AML occurrence. BAX-248A allele had shown decreased risk for AML. The combined analysis had shown that BCL2-938CA+AA-BAX-248GG group had a 1.63-fold (95 % CI: 1.08-2.45, p = 0.02) increased risk for AML. None of the clinical variables had shown any significant association with both polymorphisms. With respect to complete remission (CR) rate, BAX-248GG genotype (p = 0.002) and G allele (p = 0.009) had conferred significant risk for complete remission failure. Although the log rank test was not significant, survival analysis had shown a trend where BCL2-938CA genotype, and BAX-248GG had reduced median disease-free survival (DFS) of 9 and 10 months, respectively. In conclusion, BCL2-938C>A and BAX-248G>A gene polymorphisms might contribute to the origin of AML. Moreover, influence of BAX-248GG genotype on CR and DFS rate suggests that the BAX-248G>A polymorphism can serve as

  5. Derivation of human embryonic stem cells in defined conditions.

    Science.gov (United States)

    Ludwig, Tenneille E; Levenstein, Mark E; Jones, Jeffrey M; Berggren, W Travis; Mitchen, Erika R; Frane, Jennifer L; Crandall, Leann J; Daigh, Christine A; Conard, Kevin R; Piekarczyk, Marian S; Llanas, Rachel A; Thomson, James A

    2006-02-01

    We have previously reported that high concentrations of basic fibroblast growth factor (bFGF) support feeder-independent growth of human embryonic stem (ES) cells, but those conditions included poorly defined serum and matrix components. Here we report feeder-independent human ES cell culture that includes protein components solely derived from recombinant sources or purified from human material. We describe the derivation of two new human ES cell lines in these defined culture conditions.

  6. TET2 exon 2 skipping is an independent favorable prognostic factor for cytogenetically normal acute myelogenous leukemia (AML): TET2 exon 2 skipping in AML.

    Science.gov (United States)

    Mohamed, Aminetou Mint; Balsat, Marie; Koering, Catherine; Maucort-Boulch, Delphine; Boissel, Nicolas; Payen-Gay, Lea; Cheok, Meyling; Mortada, Hussein; Auboeuf, Didier; Pinatel, Christiane; El-Hamri, Mohamed; Tigaud, Isabelle; Hayette, Sandrine; Dumontet, Charles; Cros, Emeline; Flandrin-Gresta, Pascale; Nibourel, Olivier; Preudhomme, Claude; Thomas, Xavier; Nicolini, Franck-Emmanuel; Solly, Françoise; Guyotat, Denis; Campos, Lydia; Michallet, Mauricette; Ceraulo, Antony; Mortreux, Franck; Wattel, Eric

    2017-01-16

    In AML, approximately one-third of expressed genes are abnormally spliced, including aberrant TET2 exon 2 expression. In a discovery cohort (n=99), TET2 exon 2 skipping (TET2E2S) was found positively associated with a significant reduction in the cumulative incidence of relapse (CIR). Age, cytogenetics, and TET2E2S were independent prognostic factors for disease-free survival (DFS), and favorable effects on outcomes predominated in cytogenetic normal (CN)-AML and younger patients. Using the same cutoff in a validation cohort of 86 CN-AML patients, TET2E2S(high) patients were found to be younger than TET2(low) patients without a difference in the rate of complete remission. However, TET2E2S(high) patients exhibited a significantly lower CIR (p<10(-4)). TET2E2S and FLT3-ITD, but not age or NPM1 mutation status were independent prognostic factors for DFS and event-free survival (EFS), while TET2E2S was the sole prognostic factor that we identified for overall survival (OS). In both the intermediate-1 and favorable ELN genetic categories, TET2E2S remained significantly associated with prolonged survival. There was no correlation between TET2E2S status and outcomes in 34 additional AML patients who were unfit for IC. Therefore our results suggest that assessments of TET2 exon 2 splicing status might improve risk stratification in CN-AML patients treated with IC.

  7. Introduction: characterization and functions of human T regulatory cells.

    Science.gov (United States)

    Romagnani, Sergio

    2005-06-01

    The field of human T regulatory (Treg) cells is a rapidly progressing, but still confused field of immunology. The effects of dendritic cell (DC) manipulation in Treg generation and the main features of human "natural" Treg cells, as well as of different populations of adaptive Treg subsets, are still partially unclear. However, it is clear that Treg cells play an important role in human diseases, such as autoimmune disorders, allergy, HIV infection, tumors and graft-versus-host disease.

  8. Human pancreatic cell autotransplantation following total pancreatectomy.

    Science.gov (United States)

    Traverso, L W; Abou-Zamzam, A M; Longmire, W P

    1981-01-01

    During total pancreaticoduodenectomy for chronic pancreatitis, four patients received an intraportal pancreatic mixed-cell autograft prepared by collagenase digestion. The technique of this autotransplantation procedure was successfully developed using a normal canine pancreas, but has proved difficult to apply in the human chronic pancreatitis model. Our four patients became insulin-dependent, with proof of intrahepatic insulin production in only one patient. Three factors have contributed to the lack of graft success: 1) the preoperative endocrine status, 2) systemic hypotension and portal hypertension secondary to graft infusion, and 3) difficulty applying the successful technique in a normal dog pancreas to an extensively scarred human pancreas. The preoperative insulin response during a glucose tolerance test was blunted or delayed in the three patients tested. An immediate decrease in blood pressure and rise in portal pressure occurred in every patient and prevented infusion of the entire graft (30-50%) in three patients. Unfortunately, the patient with the most compromised insulin status was the only patient able to receive the entire graft. Our experience would indicate that further refinements in technique are necessary to prevent the vascular reaction and allow infusion of the entire graft. Furthermore, normal islet cell function is necessary before a successful graft can be expected. PMID:6781424

  9. Sourcing human embryos for embryonic stem cell lines: Problems & perspectives

    OpenAIRE

    Mehta, Rajvi H.

    2014-01-01

    The ability to successfully derive human embryonic stem cells (hESC) lines from human embryos following in vitro fertilization (IVF) opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been ′discarded′ or ′spare′ fresh or frozen human embryos following IVF...

  10. Efficient Induction and Isolation of Human Primordial Germ Cell-Like Cells from Competent Human Pluripotent Stem Cells.

    Science.gov (United States)

    Irie, Naoko; Surani, M Azim

    2017-01-01

    We recently reported a robust and defined culture system for the specification of human primordial germ cell-like cells (hPGCLCs) from human pluripotent stem cells (hPSCs), both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in vitro (Irie et al. Cell 160: 253-268, 2015). Similar attempts previously produced hPGCLCs from hPSCs at a very low efficiency, and the resulting cells were not fully characterized. A key step, which facilitated efficient hPGCLC specification from hPSCs, was the induction of a "competent" state for PGC fate via the medium containing a cocktail of four inhibitors. The competency of hPSCs can be maintained indefinitely and interchangeably with the conventional/low-competent hPSCs. Specification of hPGCLC occurs following sequential expression of key germ cell fate regulators, notably SOX17 and BLIMP1, as well as initiation of epigenetic resetting over 5 days. The hPGCLCs can be isolated using specific cell surface markers without the need for generating germ cell-specific reporter hPSC lines. This powerful method for the induction and isolation of hPGCLCs can be applied to both hESCs and iPSCs, which can be used for advances in human germ line biology.

  11. The similarity between human embryonic stem cell-derived epithelial cells and ameloblast-lineage cells

    Institute of Scientific and Technical Information of China (English)

    Li-Wei Zheng; Logan Linthicum; Pamela K DenBesten; Yan Zhang

    2013-01-01

    This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCI) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which ,vas also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel.

  12. Radiosensitivity of Human Melanoma Cell Lines

    Energy Technology Data Exchange (ETDEWEB)

    Bergoc, R. M.; Medina, V.; Cricco, G.; Mohamed, N.; Martin, G.; Nunez, M.; Croci, M.; Crescenti, E. J.; Rivera, E. S.

    2004-07-01

    Cutaneous melanoma is a skin cancer resulting from the malign transformation of skin-pigment cells, the melanocytes. The radiotherapy, alone or in combination with other treatment, is an important therapy for this disease. the objective of this paper was to determine in vitro the radiosensitivity of two human melanoma cell lines with different metastatic capability: WM35 and MI/15, and to study the effect of drugs on radiobiological parameters. The Survival Curves were adjusted to the mathematical Linear-quadratic model using GrapsPad Prism software. Cells were seeded in RPMI medium (3000-3500 cells/flask), in triplicate and irradiated 24 h later. The irradiation was performed using an IBL 437C H Type equipment (189 TBq, 7.7 Gy/min) calibrated with a TLD 700 dosimeter. The range of Doses covered from 0 to 10 Gy and the colonies formed were counted at day 7th post-irradiation. Results obtained were: for WM35, {alpha}=0.37{+-}0.07 Gy''-1 and {beta}=0.06{+-}0.02 Gy''-2, for M1/15m {alpha}=0.47{+-}0.03 Gy''-1 and {beta}=0.06{+-}0.01 Gy''-2. The {alpha}/{beta} values WM35: {alpha}/{beta} values WM35: {alpha}/{beta}=6.07 Gy and M1/15: {alpha}/{beta}{sub 7}.33 Gy were similar, independently of their metastatic capabillity and indicate that both lines exhibit high radioresistance. Microscopic observation of irradiated cells showed multinuclear cells with few morphologic changes non-compatible with apoptosis. By means of specific fluorescent dyes and flow cytometry analysis we determined the intracellular levels of the radicals superoxide and hydrogen peroxide and their modulation in response to ionizing radiation. The results showed a marked decreased in H{sub 2}O{sub 2} intracellular levels with a simultaneous increase in superoxide that will be part of a mechanism responsible for induction of cell radioresistance. This response triggered by irradiated cells could not be abrogated by different treatments like histamine or the

  13. Depletion of endogenous tumor-associated regulatory T cells improves the efficacy of adoptive cytotoxic T-cell immunotherapy in murine acute myeloid leukemia.

    Science.gov (United States)

    Zhou, Qing; Bucher, Christoph; Munger, Meghan E; Highfill, Steven L; Tolar, Jakub; Munn, David H; Levine, Bruce L; Riddle, Megan; June, Carl H; Vallera, Daniel A; Weigel, Brenda J; Blazar, Bruce R

    2009-10-29

    Tumor-induced immune suppression can permit tumor cells to escape host immune resistance. To elucidate host factors contributing to the poor response of adoptively transferred tumor-reactive cytotoxic T lymphocytes (CTLs), we used a systemic model of murine acute myeloid leukemia (AML). AML progression resulted in a progressive regulatory T-cell (Treg) accumulation in disease sites. The adoptive transfer of in vitro-generated, potently lytic anti-AML-reactive CTLs failed to reduce disease burden or extend survival. Compared with non-AML-bearing hosts, transferred CTLs had reduced proliferation in AML sites of metastases. Treg depletion by a brief course of interleukin-2 diphtheria toxin (IL-2DT) transiently reduced AML disease burden but did not permit long-term survival. In contrast, IL-2DT prevented anti-AML CTL hypoproliferation, increased the number of transferred CTLs at AML disease sites, reduced AML tumor burden, and resulted in long-term survivors that sustained an anti-AML memory response. These data demonstrated that Tregs present at AML disease sites suppress adoptively transferred CTL proliferation, limiting their in vivo expansion, and Treg depletion before CTL transfer can result in therapeutic efficacy in settings of substantial pre-existing tumor burden in which antitumor reactive CTL infusion alone has proven ineffective.

  14. Generation of human induced pluripotent stem cells from dermal fibroblasts.

    Science.gov (United States)

    Lowry, W E; Richter, L; Yachechko, R; Pyle, A D; Tchieu, J; Sridharan, R; Clark, A T; Plath, K

    2008-02-26

    The generation of patient-specific pluripotent stem cells has the potential to accelerate the implementation of stem cells for clinical treatment of degenerative diseases. Technologies including somatic cell nuclear transfer and cell fusion might generate such cells but are hindered by issues that might prevent them from being used clinically. Here, we describe methods to use dermal fibroblasts easily obtained from an individual human to generate human induced pluripotent stem (iPS) cells by ectopic expression of the defined transcription factors KLF4, OCT4, SOX2, and C-MYC. The resultant cell lines are morphologically indistinguishable from human embryonic stem cells (HESC) generated from the inner cell mass of a human preimplantation embryo. Consistent with these observations, human iPS cells share a nearly identical gene-expression profile with two established HESC lines. Importantly, DNA fingerprinting indicates that the human iPS cells were derived from the donor material and are not a result of contamination. Karyotypic analyses demonstrate that reprogramming of human cells by defined factors does not induce, or require, chromosomal abnormalities. Finally, we provide evidence that human iPS cells can be induced to differentiate along lineages representative of the three embryonic germ layers indicating the pluripotency of these cells. Our findings are an important step toward manipulating somatic human cells to generate an unlimited supply of patient-specific pluripotent stem cells. In the future, the use of defined factors to change cell fate may be the key to routine nuclear reprogramming of human somatic cells.

  15. 15,16-Dihydrotanshinone I from the Functional Food Salvia miltiorrhiza Exhibits Anticancer Activity in Human HL-60 Leukemia Cells: in Vitro and in Vivo Studies.

    Science.gov (United States)

    Liu, Jun-Jen; Wu, Hsueh-Hsia; Chen, Tzu-Ho; Leung, Wan; Liang, Yu-Chih

    2015-08-17

    15,16-Dihydrotanshinone I (DHTS) is extracted from Salvia miltiorrhiza Bunge which is a functional food in Asia. In this study, we investigated the apoptotic effect of DHTS on the human acute myeloid leukemia (AML) type III HL-60 cell line. We found that treatment with 1.5 μg/mL DHTS increased proapoptotic Bax and Bad protein expressions and activated caspases-3, -8, and -9, thus leading to poly ADP ribose polymerase (PARP) cleavage and resulting in cell apoptosis. DHTS induced sustained c-Jun N-terminal kinase (JNK) phosphorylation and Fas ligand (FasL) expression. The anti-Fas blocking antibody reversed the DHTS-induced cell death, and the JNK-specific inhibitor, SP600125, inhibited DHTS-induced caspase-3, -8, -9, and PARP cleavage. In a xenograft nude mice model, 25 mg/kg DHTS showed a great effect in attenuating HL-60 tumor growth. Taken together, these results suggest that DHTS can induce HL-60 cell apoptosis in vitro and inhibit HL-60 cell growth in vivo; the underlying mechanisms might be mediated through activation of the JNK and FasL signal pathways.

  16. Stereological quantification of mast cells in human synovium

    DEFF Research Database (Denmark)

    Damsgaard, T E; Sørensen, Flemming Brandt; Herlin, T;

    1999-01-01

    Mast cells participate in both the acute allergic reaction as well as in chronic inflammatory diseases. Earlier studies have revealed divergent results regarding the quantification of mast cells in the human synovium. The aim of the present study was therefore to quantify these cells in the human...... synovium, using stereological techniques. Different methods of staining and quantification have previously been used for mast cell quantification in human synovium. Stereological techniques provide precise and unbiased information on the number of cell profiles in two-dimensional tissue sections of......, in this case, human synovium. In 10 patients suffering from osteoarthritis a median of 3.6 mast cells/mm2 synovial membrane was found. The total number of cells (synoviocytes, fibroblasts, lymphocytes, leukocytes) present was 395.9 cells/mm2 (median). The mast cells constituted 0.8% of all the cell profiles...

  17. Technical Challenges in the Derivation of Human Pluripotent Cells

    Directory of Open Access Journals (Sweden)

    Parinya Noisa

    2011-01-01

    Full Text Available It has long been discovered that human pluripotent cells could be isolated from the blastocyst state of embryos and called human embryonic stem cells (ESCs. These cells can be adapted and propagated indefinitely in culture in an undifferentiated manner as well as differentiated into cell representing the three major germ layers: endoderm, mesoderm, and ectoderm. However, the derivation of human pluripotent cells from donated embryos is limited and restricted by ethical concerns. Therefore, various approaches have been explored and proved their success. Human pluripotent cells can also be derived experimentally by the nuclear reprogramming of somatic cells. These techniques include somatic cell nuclear transfer (SCNT, cell fusion and overexpression of pluripotent genes. In this paper, we discuss the technical challenges of these approaches for nuclear reprogramming, involving their advantages and limitations. We will also highlight the possible applications of these techniques in the study of stem cell biology.

  18. Human B cell activating factor (BCAF): production by a human T cell tumor line.

    Science.gov (United States)

    Fevrier, M; Diu, A; Mollier, P; Abadie, A; Olive, D; Mawas, C; Theze, J

    1989-01-01

    In a previous study, we demonstrated that supernatants from human T cell clones stimulated by a pair of anti-CD2 monoclonal antibodies cause resting human B cells to become activated and to proliferate in the absence of any other signals. The activity responsible for these effects was shown to be different from already characterized lymphokines and in particular from IL-2 and IL-4, and was named B Cell Activating Factor or BCAF. In this paper, we describe the production of BCAF by a human T cell tumor line T687 after phorbol myristate acetate (PMA) stimulation; this production can be potentiated by phytohemagglutinin (PHA). We further show that the stimulatory phase can be separated from the secretory phase thereby avoiding contamination of BCAF-containing supernatant by PMA and PHA. Supernatants produced under these conditions do not contain either IL-4 or IFN but contain traces of lymphotoxin and 2 to 10 ng/ml of IL-2. The T687 cell line will allow us to obtain a large volume of supernatant for biochemical study and purification of the molecule(s) responsible for BCAF activity.

  19. Trichloroethylene toxicity in a human hepatoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Thevenin, E.; McMillian, J. [Medical Univ. of Charleston South Carolina, SC (United States)

    1994-12-31

    The experiments conducted in this study were designed to determine the usefullness of hepatocyte cultures and a human hepatoma cell line as model systems for assessing human susceptibility to hepatocellular carcinoma due to exposure to trichloroethylene. The results from these studies will then be analyzed to determine if human cell lines can be used to conduct future experiments of this nature.

  20. Human Immunodeficiency Syndromes Affecting Human Natural Killer Cell Cytolytic Activity

    OpenAIRE

    Ham, Hyoungjun; Billadeau, Daniel D.

    2014-01-01

    Natural killer (NK) cells are lymphocytes of the innate immune system that secrete cytokines upon activation and mediate the killing of tumor cells and virus-infected cells, especially those that escape the adaptive T cell response caused by the down regulation of MHC-I. The induction of cytotoxicity requires that NK cells contact target cells through adhesion receptors, and initiate activation signaling leading to increased adhesion and accumulation of F-actin at the NK cell cytotoxic synaps...

  1. Human immunodeficiency virus type 1 infection of human uterine epithelial cells: viral shedding and cell contact-mediated infectivity.

    Science.gov (United States)

    Asin, Susana N; Wildt-Perinic, Dunja; Mason, Sarah I; Howell, Alexandra L; Wira, Charles R; Fanger, Michael W

    2003-05-15

    We examined the mechanism of human immunodeficiency virus (HIV) type 1 infection of human uterine epithelial cells to gain a clearer understanding of the events by which HIV-1 infects cells within the female reproductive tract. We demonstrated that these cells can be productively infected by HIV-1 and that infection is associated with viral RNA reverse transcription, DNA transcription, and secretion of infectious virus. Levels of viral DNA and secreted virus decreased gradually after infection. Moreover, virus released by the uterine epithelial cells shortly after infection was able to infect human T cell lines, but virus released later did not. In contrast, human CD4(+) T cell lines were infected after cocultivation with epithelial cells at both early and late stages of infection. These data demonstrated that HIV-1 infects human epithelial cells of upper reproductive tract origin and that productive viral infection of epithelial cells may be an important mechanism of transmission of HIV-1 infection in women.

  2. Effect of human neural progenitor cells on injured spinal cord

    Institute of Scientific and Technical Information of China (English)

    XU Guang-hui; BAI Jin-zhu; CAI Qin-lin; LI Xiao-xia; LI Ling-song; SHEN Li

    2005-01-01

    Objective: To study whether human neural progenitor cells can differentiate into neural cells in vivo and improve the recovery of injured spinal cord in rats.Methods: Human neural progenitor cells were transplanted into the injured spinal cord and the functional recovery of the rats with spinal cord contusion injury was evaluated with Basso-Beattie-Bresnahan (BBB) locomotor scale and motor evoked potentials. Additionally, the differentiation of human neural progenitor cells was shown by immunocytochemistry.Results: Human neural progenitor cells developed into functional cells in the injured spinal cord and improved the recovery of injured spinal cord in both locomotor scores and electrophysiological parameters in rats.Conclusions: Human neural progenitor cells can treat injured spinal cord, which may provide a new cell source for research of clinical application.

  3. Generation of induced pluripotent stem cells from human blood.

    Science.gov (United States)

    Loh, Yuin-Han; Agarwal, Suneet; Park, In-Hyun; Urbach, Achia; Huo, Hongguang; Heffner, Garrett C; Kim, Kitai; Miller, Justine D; Ng, Kitwa; Daley, George Q

    2009-05-28

    Human dermal fibroblasts obtained by skin biopsy can be reprogrammed directly to pluripotency by the ectopic expression of defined transcription factors. Here, we describe the derivation of induced pluripotent stem cells from CD34+ mobilized human peripheral blood cells using retroviral transduction of OCT4/SOX2/KLF4/MYC. Blood-derived human induced pluripotent stem cells are indistinguishable from human embryonic stem cells with respect to morphology, expression of surface antigens, and pluripotency-associated transcription factors, DNA methylation status at pluripotent cell-specific genes, and the capacity to differentiate in vitro and in teratomas. The ability to reprogram cells from human blood will allow the generation of patient-specific stem cells for diseases in which the disease-causing somatic mutations are restricted to cells of the hematopoietic lineage.

  4. A Chemical Probe that Labels Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Nao Hirata

    2014-03-01

    Full Text Available A small-molecule fluorescent probe specific for human pluripotent stem cells would serve as a useful tool for basic cell biology research and stem cell therapy. Screening of fluorescent chemical libraries with human induced pluripotent stem cells (iPSCs and subsequent evaluation of hit molecules identified a fluorescent compound (Kyoto probe 1 [KP-1] that selectively labels human pluripotent stem cells. Our analyses indicated that the selectivity results primarily from a distinct expression pattern of ABC transporters in human pluripotent stem cells and from the transporter selectivity of KP-1. Expression of ABCB1 (MDR1 and ABCG2 (BCRP, both of which cause the efflux of KP-1, is repressed in human pluripotent stem cells. Although KP-1, like other pluripotent markers, is not absolutely specific for pluripotent stem cells, the identified chemical probe may be used in conjunction with other reagents.

  5. Enhanced casein kinase II activity in human tumour cell cultures

    DEFF Research Database (Denmark)

    Prowald, K; Fischer, H; Issinger, O G

    1984-01-01

    Casein kinase II (CKII) activity is enhanced as much as 2-3 fold in established and 4-5-fold in transformed human cell lines when compared to that of fibroblasts and primary human tumour cell cultures where CKII activity never exceeded a basic level. The high activity of CKII in transformed cells...

  6. MODERATE CYTOTOXICITY OF PROANTHOCYANIDINS TO HUMAN TUMOR-CELL LINES

    NARCIS (Netherlands)

    KOLODZIEJ, H; HABERLAND, C; WOERDENBAG, HJ; KONINGS, AWT

    1995-01-01

    In the present study the cytotoxicity of 16 proanthocyanidins was evaluated in GLC(4), a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. With IC50 values ranging from 18 to >200 mu m following continuous

  7. Derivation and differentiation of haploid human embryonic stem cells.

    Science.gov (United States)

    Sagi, Ido; Chia, Gloryn; Golan-Lev, Tamar; Peretz, Mordecai; Weissbein, Uri; Sui, Lina; Sauer, Mark V; Yanuka, Ofra; Egli, Dieter; Benvenisty, Nissim

    2016-04-07

    Diploidy is a fundamental genetic feature in mammals, in which haploid cells normally arise only as post-meiotic germ cells that serve to ensure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species, but haploid human ES cells have yet to be reported. Here we generated and analysed a collection of human parthenogenetic ES cell lines originating from haploid oocytes, leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics, such as self-renewal capacity and a pluripotency-specific molecular signature. Moreover, we demonstrated the utility of these cells as a platform for loss-of-function genetic screening. Although haploid human ES cells resembled their diploid counterparts, they also displayed distinct properties including differential regulation of X chromosome inactivation and of genes involved in oxidative phosphorylation, alongside reduction in absolute gene expression levels and cell size. Surprisingly, we found that a haploid human genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers both in vitro and in vivo, despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics and development.

  8. Human embryonic stem cells and respect for life

    OpenAIRE

    Meyer, J.(CERN, Geneva, Switzerland)

    2000-01-01

    The purpose of this essay is to stimulate academic discussion about the ethical justification of using human primordial stem cells for tissue transplantation, cell replacement, and gene therapy. There are intriguing alternatives to using embryos obtained from elective abortions and in vitro fertilisation to reconstitute damaged or dysfunctional human organs. These include the expansion and transplantation of latent adult progenitor cells.

  9. The Broad Anti-AML Activity of the CD33/CD3 BiTE Antibody Construct, AMG 330, Is Impacted by Disease Stage and Risk.

    Science.gov (United States)

    Harrington, Kimberly H; Gudgeon, Chelsea J; Laszlo, George S; Newhall, Kathryn J; Sinclair, Angus M; Frankel, Stanley R; Kischel, Roman; Chen, Guang; Walter, Roland B

    2015-01-01

    The CD33/CD3-bispecific T-cell engaging (BiTE) antibody construct, AMG 330, potently lyses CD33+ leukemic cells in vitro. Using specimens from 41 patients with acute myeloid leukemia (AML), we studied the factors that might contribute to clinical response or resistance. For this purpose, thawed aliquots of primary AML samples were immunophenotypically characterized and subjected to various doses of AMG 330 in the presence or absence of healthy donor T-cells. After 48 hours, drug-specific cytotoxicity was quantified and correlated with CD33 expression levels, amounts of T-cells present, and other disease characteristics. AMG 330 caused modest cytotoxicity that was correlated with the amount of autologous T-cells (P = 0.0001) but not CD33 expression, as AMG 330 exerted marked cytotoxic effects in several specimens with minimal CD33 expression. With healthy donor T-cells added, AMG 330 cytotoxicity depended on the drug dose and effector:target (E:T) cell ratio. High cytotoxic activity was observed even with minimal CD33 expression, and AMG 330 cytotoxicity and CD33 expression correlated only at high E:T cell ratio and high AMG 330 doses (Pspectrum, while suggesting the presence of yet undefined, CD33-independent, relative resistance mechanisms in specific patient subsets.

  10. Abnormalities in human pluripotent cells due to reprogramming mechanisms.

    Science.gov (United States)

    Ma, Hong; Morey, Robert; O'Neil, Ryan C; He, Yupeng; Daughtry, Brittany; Schultz, Matthew D; Hariharan, Manoj; Nery, Joseph R; Castanon, Rosa; Sabatini, Karen; Thiagarajan, Rathi D; Tachibana, Masahito; Kang, Eunju; Tippner-Hedges, Rebecca; Ahmed, Riffat; Gutierrez, Nuria Marti; Van Dyken, Crystal; Polat, Alim; Sugawara, Atsushi; Sparman, Michelle; Gokhale, Sumita; Amato, Paula; Wolf, Don P; Ecker, Joseph R; Laurent, Louise C; Mitalipov, Shoukhrat

    2014-07-10

    Human pluripotent stem cells hold potential for regenerative medicine, but available cell types have significant limitations. Although embryonic stem cells (ES cells) from in vitro fertilized embryos (IVF ES cells) represent the 'gold standard', they are allogeneic to patients. Autologous induced pluripotent stem cells (iPS cells) are prone to epigenetic and transcriptional aberrations. To determine whether such abnormalities are intrinsic to somatic cell reprogramming or secondary to the reprogramming method, genetically matched sets of human IVF ES cells, iPS cells and nuclear transfer ES cells (NT ES cells) derived by somatic cell nuclear transfer (SCNT) were subjected to genome-wide analyses. Both NT ES cells and iPS cells deri