scholarly journals Aberrant Overexpression of the Wilms Tumor Gene (WT1) in Human Leukemia

Blood ◽  
1997 ◽  
Vol 89 (4) ◽  
pp. 1405-1412 ◽  
Author(s):  
Kazushi Inoue ◽  
Hiroyasu Ogawa ◽  
Yoshiaki Sonoda ◽  
Takafumi Kimura ◽  
Hideaki Sakabe ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Wilms Tumor ◽  
Leukemic Cells ◽  
Hematopoietic Progenitor Cells ◽  
Cell Populations ◽  
Human Leukemia ◽  
Wt1 Gene ◽  
Hematopoietic Progenitor ◽  
Expression Levels ◽  
Hla Dr

Abstract To clarify whether the expression of the WT1 gene in leukemic cells is aberrant or merely reflects that in normal counterparts, the expression levels of the WT1 gene were quantitated for normal hematopoietic progenitor cells. Bone marrow (BM) and umbilical cord blood (CB) cells were fluorescence-activated cell sorting (FACS)-sorted into CD34+ and CD34− cell populations, and the CD34+ cells into nine subsets (CD34+CD33−, CD34+CD33+, CD34+CD38−, CD34+CD38+, CD34+HLA-DR−, CD34+HLA-DR+, CD34+c-kithigh, CD34+c-kitlow, and CD34+c-kit−) according to the expression levels of CD34, CD33, CD38, HLA-DR, and c-kit. Moreover, acute myeloid leukemic cells were also FACS-sorted into four populations (CD34+CD33−, CD34+CD33+, CD34− CD33+, and CD34− CD33−). FACS-sorted normal hematopoietic progenitor and leukemic cells and FACS-unsorted leukemic cells were examined for the WT1 expression by quantitative reverse transcriptase-polymerase chain reaction. The WT1 expression in the CD34+ and CD34− cell populations and in the nine CD34+ subsets of BM and CB was at either very low (1.0 to 2.4 × 10−2) or undetectable (<10−2) levels (the WT1 expression level of K562 cells was defined as 1.0), whereas the average levels of WT1 expression in FACS-sorted and -unsorted leukemic cells were 2.4 to 9.3 × 10−1. Thus, the WT1 expression levels in normal hematopoietic progenitor cells were at least 10 times less than those in leukemic cells. Therefore, we could not find any normal counterparts of BM or CB that expressed the WT1 at levels comparable with those in leukemic cells. These results indicate an aberrant overexpression of the WT1 gene in leukemic cells and imply the involvement of this gene in human leukemogenesis.

scholarly journals Wilms' Tumor Gene (WT1) Competes With Differentiation-Inducing Signal in Hematopoietic Progenitor Cells

Blood ◽  
1998 ◽  
Vol 91 (8) ◽  
pp. 2969-2976 ◽  
Author(s):  
Kazushi Inoue ◽  
Hiroya Tamaki ◽  
Hiroyasu Ogawa ◽  
Yoshihiro Oka ◽  
Toshihiro Soma ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Tumor Suppressor ◽  
Progenitor Cells ◽  
Wilms Tumor ◽  
Suppressor Gene ◽  
Hematopoietic Progenitor Cells ◽  
Wt1 Gene ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Oncogenic Function

The WT1 gene is a tumor-suppressor gene that was isolated as a gene responsible for Wilms' tumor, a childhood kidney neoplasm. We have previously reported that the WT1 gene is strongly expressed in leukemia cells with an increase in its expression levels at relapse and an inverse correlation between its expression levels and prognosis, thus making it a novel tumor marker for leukemic blast cells. Furthermore, WT1 antisense oligomers have been found to inhibit the growth of leukemic cells. These results strongly suggested the involvement of the WT1 gene in human leukemogenesis. The present study was performed to prove our hypothesis that the WT1 gene plays a key role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells, rather than a tumor-suppressor gene function. 32D cl3, an interleukin-3–dependent myeloid progenitor cell line, differentiates into mature neutrophils in response to granulocyte colony-stimulating factor (G-CSF). However, when transfected wild-type WT1 gene was constitutively expressed in 32D cl3, the cells stopped differentiating and continued to proliferate in response to G-CSF. As for signal transduction mediated by G-CSF receptor (G-CSFR), Stat3α was constitutively activated in wild-type WT1-infected 32D cl3 in response to G-CSF, whereas, in WT1-uninfected 32D cl3, activation of Stat3α was only transient. However, most interesting was the fact that G-CSF stimulation resulted in constitutive activation of Stat3β only in wild-type WT1-infected 32D cl3, but not in WT1-uninfected 32D cl3. Thus, WT1 expression constitutively activated both Stat3α and Stat3β. A transient activation of Stat1 was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation, but no difference in its activation was found. No activation of MAP kinase was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation. These results demonstrated that WT1 expression competed with the differentiation-inducing signal mediated by G-CSFR and constitutively activated Stat3, resulting in the blocking of differentiation and subsequent proliferation. Therefore, the data presented here support our hypothesis that the WT1 gene plays an essential role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells and represent the first demonstration of an important role of the WT1 gene in signal transduction in hematopoietic progenitor cells.

scholarly journals Long-term generation and expansion of human primitive hematopoietic progenitor cells in vitro

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 661-669 ◽  
Author(s):  
EF Srour ◽  
JE Brandt ◽  
RA Briddell ◽  
S Grigsby ◽  
T Leemhuis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Fold Increase ◽  
Hematopoietic Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Progenitor ◽  
Hla Dr

Abstract Although sustained production of committed human hematopoietic progenitor cells in long-term bone marrow cultures (LTBMC) is well documented, evidence for the generation and expansion of human primitive hematopoietic progenitor cells (PHPC) in such cultures is lacking. For that purpose, we attempted to determine if the human high proliferative potential colony-forming cell (HPP-CFC), a primitive hematopoietic marrow progenitor cell, is capable of generation and expansion in vitro. To that effect, stromal cell-free LTBMC were initiated with CD34+ HLA-DR-CD15- rhodamine 123dull bone marrow cells and were maintained with repeated addition of c-kit ligand and a synthetic interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein. By day 21 of LTBMC, a greater than twofold increase in the number of assayable HPP-CFC was detected. Furthermore, the production of HPP-CFC in LTBMC continued for up to 4 weeks, resulting in a 5.5-fold increase in HPP-CFC numbers. Weekly phenotypic analyses of cells harvested from LTBMC showed that the number of CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 x 10(4) by day 21. To examine further the nature of the in vitro HPP-CFC expansion, individual HPP- CFC colonies were serially cloned. Secondary cloning of individual, day 28 primary HPP-CFC indicated that 46% of these colonies formed an average of nine secondary colony-forming unit--granulocyte-macrophage (CFU-GM)--derived colonies, whereas 43% of primary HPP-CFC gave rise to between one and six secondary HPP-CFC colonies and 6 to 26 CFU-GM. These data show that CD34+ HLA-DR- CD15- rhodamine 123dull cells represent a fraction of human bone marrow highly enriched for HPP-CFC and that based on their regeneration and proliferative capacities, a hierarchy of HPP-CFC exists. Furthermore, these studies indicate that in the presence of appropriate cytokine stimulation, it is possible to expand the number of PHPC in vitro.

Understanding The Marrow Niche: Advanced 3D Model System Allows Functional Analysis Of The Interaction With Human Hematopoietic Progenitor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2462-2462
Author(s):  
Patrick Wuchter ◽  
Rainer Saffrich ◽  
Stefan Giselbrecht ◽  
Patrick Horn ◽  
Anthony D. Ho ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Model System ◽  
Hematopoietic Progenitor Cells ◽  
Cell Populations ◽  
Colony Formation Assay ◽  
Hematopoietic Progenitor ◽  
Hypoxic Conditions ◽  
The Impact

Abstract We previously demonstrated that “stemness” of human hematopoietic progenitor cells (HPC) was maintained in a co-culture setting with a monolayer of human mesenchymal stromal cells (MSC). To simulate and monitor the marrow microenvironment of the HPC niche more precisely we have established a 3D co-culture system based on a proprietary KITChip. The KITChip was developed by the Karlsruhe Institute of Technology (KIT) and represents a unique microchip with defined microwell cavities for 3D cell cultures. Sample acquisition was approved by the local Ethics Committee and informed written consent was obtained from all subjects. MSC were derived from human bone marrow of healthy voluntary donors, and HPC were isolated from umbilical cord blood. Cells were mixed in suspension in a ratio of 3:2 (3x105 MSC and 2x105 HPC) and inoculated into the KITChip, which was subsequently mounted into a microbioreactor. This closed loop setup allowed precise control of medium flow and oxygen saturation. After 1 to 5 days of co-culture, the two cell populations were analyzed by immunostaining, RT2-PCR and colony formation assay. MSC form a complex 3D mesh in the microcavities of the KITChip and were maintained stable for up to 6 weeks. We have demonstrated that HPC were distributed three-dimensionally inside this MSC mesh and could be kept viable in this environment for at least 14 days. A defined proportion of CD34+ HPC adhered to the MSC in the microcavities and built up direct cellular connections to the surrounding MSC. By means of RT2-PCR, we could demonstrate that throughout the whole culture period of 14 days a subpopulation of CD34+/p21+/CXCR4+ cells was maintained in the 3D-environment more efficiently than compared to conventional co-culture with MSC monolayer. This was confirmed by Western blotting after the isolation of both cell populations from the chip. The colony formation assay revealed that the plasticity of the HPC cultivated in the 3D KITChip was nearly the same as that of freshly isolated HPC at day 0, whereas HPC co-cultured on MSC monolayer showed a significant decrease in stem cell plasticity. Further analysis under hypoxic conditions (5% O2) indicated that gene expressions of CD33, CD34, CD38 and CD44 were markedly reduced, while those of CD90, CD105, c-Kit, p21, SDF-1 and Angpt-1 remained stable compared to normoxic culture conditions. This novel model system allows analysis of the major determinants of the niche and the impact of a 3D microenvironment on vital stem cell functions. Early HPC were maintained more efficiently and showed a superior plasticity potential when cultured in the 3D KITChip as compared to conventional 2D co-culture systems. Current studies are in process to define the functional significance of the observed changes in gene expression pattern under hypoxic conditions, which resembles the physiologic milieu of the marrow. Disclosures: Wuchter: ETICHO: Consultancy, Honoraria; Sanofi: Honoraria for lectures Other. Ho:Sanofi-Genzyme: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Cannabinoids Derivatives Exert a Potent Antileukemic Effect By Modifying the Pattern of Membrane Sphingolipids

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4718-4718
Author(s):  
Mayte Medrano ◽  
María Victoria Barbado-Gonzalez ◽  
Nuria Campillo ◽  
Francisco Hidalgo ◽  
Teresa Caballero-Velazquez ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Leukemic Cells ◽  
Hematopoietic Progenitor Cells ◽  
Cb2 Receptor ◽  
Hematopoietic Progenitor ◽  
Healthy Donors ◽  
Win 55

Abstract Endocannabinoid system is a set of ligands, receptors and endogenous enzymes which modulate a variety of physiological effects. There are two well-characterized cannabinoid receptors, CB1 (mainly expressed in Central Nervous System) and CB2 (mainly in hematopoietic cells). Here, we tested the effect of the cannabinoid WIN-55 212-2 in acute myeloid leukemia (AML) in vitro, ex vivo and in vivo and studied the molecular signaling pathways involved in this effect. Moreover, we synthesized a new family of twelve cannabinoids that are specific to CB2 receptor. For their design and synthesis, computational techniques of docking, analytical and spectroscopic techniques such as mass spectrometry (MS) were used. To assess the anti-leukemia effect of the different cannabinoids, we analyzed cell viability by MTT and flow cytometry using six human AML cell lines, primary cells from healthy donors (hematopoietic progenitor cells (HPC) and lymphocytes) and blasts from AML patients. Cannabinoids induced a potent proapoptotic effect on AML cell lines and on primary leukemic cells, which was not observed in normal HPC and lymphocytes from healthy donors. Fragmentation of PARP and activation of caspases 2, 3, 8 and 9 were confirmed by western-blot. Other proteins involved in the effect of cannabinoids were p-AKT, p-ERK 1/2, p-38 and p- JNK. Also studies on p-PERK, p-IRE1 and CHOP confirmed an increased endoplasmic reticulum stress upon exposure to cannabinoids. Mitochondrial damage was analyzed by flow cytometry using TMRE and by MitoSOXTMRed. These assays confirmed a very early mitochondrial damage in leukemic cells which was not observed in normal hematopoietic progenitor cells. Moreover, we analyzed the ceramide levels, a membrane lipid associated with death/survival cell processes by HPLC and immunohistochemistry. Remarkably, we observed significant differences in the amounts of certain subtypes of ceramides in untreated versus treated leukemic cells. The proapoptotic effect of cannabinoids on AML cells was abolished upon co-culture with either CB2 receptor antagonists or with pancaspase inhibitors. Finally, NOD/scid/IL-2R gammae null (NSG) mice were xenotransplanted with HL60 cell line. We confirmed disease infiltration in bone marrow (BM) by BM aspirates and flow cytometry assays. Once the presence of leukemic cells was confirmed, treatment with vehicle, WIN-55 cannabinoid at a dose of 5 mg/kg/day or citarabine (ARA-C) at 50 mg/kg during 5 days was administered. We observed a significantly increased survival among mice treated with WIN-55 cannabinoid as compared to both the control group and the group treated with ARA-C. In addition, we tested in vivo the effect of these compounds on normal hematopoiesis by treating healthy BALB-C mice. We confirmed that cannabinoids did not affect the viability of the different populations of hematopoietic progenitors (LK, GMP, CMP) and, moreover, an increased platelet count was observed in treated mice. Our findings indicate that cannabinoids display a highly selective proapoptotic effect against leukemic cells. Several pathways are involved in this effect, the modification in the ceramide pattern playing a main role. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Tie receptor tyrosine kinase is expressed by human hematopoietic progenitor cells and by a subset of megakaryocytic cells

Blood ◽  
1996 ◽  
Vol 87 (6) ◽  
pp. 2212-2220 ◽  
Author(s):  
P Batard ◽  
P Sansilvestri ◽  
C Scheinecker ◽  
W Knapp ◽  
N Debili ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Progenitor Cells ◽  
Receptor Tyrosine Kinase ◽  
Leukemia Cell ◽  
Hematopoietic Progenitor Cells ◽  
Human Leukemia ◽  
Mouse Development ◽  
Hematopoietic Progenitor ◽  
Megakaryocytic Differentiation ◽  
Cd34 Cells

Growth factor receptors in human hematopoietic progenitor cells have become the focus of intense interest, because they may provide tools for the monitoring, enrichment, and expansion of stem cells. We have shown earlier that the Tie receptor tyrosine kinase is expressed in erythroid and megakaryoblastic human leukemia cell lines, in the blood islands of the yolk sac, and in endothelial cells starting from day 8.0 of mouse development. Here, the expression of Tie was studied in human hematopoietic cells of various sources. Peripheral blood mononuclear cells were Tie-. However, a large fraction of CD34+ cells from umbilical cord blood (UCB) and bone marrow (BM) expressed tie protein and mRNA. On average, 64% of the fluorescence-activated cell sorting- gated UCB CD34+ cells including CD38- cells and a fraction of cells expressing low levels of c-Kit were Tie+. Also, 30% to 60% of BM CD34+ cells were Tie+, including most of the BM CD34+CD38-, CD34+Thy-1+, and CD34+HLA-DR- cells. Under culture conditions allowing myeloid, erythroid, and/or megakaryocytic differentiation, purified UCB CD34+ cells lost Tie mRNA and protein expression concomitantly with that of CD34; however, a significant fraction of cells expressed Tie during megakaryocytic differentiation. These data suggest that, in humans, the Tie receptor and presumably its ligand may function at an early stage of hematopoietic cell differentiation.

scholarly journals Growth inhibition of clonogenic leukemic precursor cells by minor histocompatibility antigen-specific cytotoxic T lymphocytes.

1991 ◽  
Vol 174 (1) ◽  
pp. 27-33 ◽  
Author(s):  
J H Falkenburg ◽  
H M Goselink ◽  
D van der Harst ◽  
S A van Luxemburg-Heijs ◽  
Y M Kooy-Winkelaar ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Progenitor Cells ◽  
Cytotoxic T Lymphocytes ◽  
Specific Antigen ◽  
Precursor Cells ◽  
Leukemic Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor

Minor histocompatibility (mH) antigens appear to play a major role in bone marrow transplantation (BMT) using HLA-identical donors. Previously, we reported the isolation of major histocompatibility complex (MHC)-restricted mH antigen-specific cytotoxic T lymphocytes (CTL) from patients with graft-vs.-host disease or rejection after HLA-identical BMT. We have demonstrated that mH antigens can be recognized on hematopoietic progenitor cells, and residual recipient CTL specific for mH antigens expressed on donor hematopoietic progenitor cells may be responsible for graft rejection in spite of intensive conditioning regimens in HLA-identical BMT. Here, we investigated whether mH antigen-specific CTL directed against the mH antigens HA-1 to HA-5 and the male-specific antigen H-Y were capable of antigen-specific inhibition of in vitro growth of clonogenic leukemic precursor cells. We demonstrate that mH antigen-specific CTL against all mH antigens tested can lyse freshly obtained myeloid leukemic cells, that these mH antigen-specific CTL can inhibit their clonogenic leukemic growth in vitro, and that this recognition is MHC restricted. We illustrate that leukemic (precursor) cells can escape elimination by mH antigen-specific CTL by impaired expression of the relevant MHC restriction molecule. We suggest that mH antigen-specific MHC-restricted CTL may be involved in vivo in the graft-vs.-leukemia reactivity after BMT.

Induction of NK Cell Reactivity Against Myeloid Leukemia By a Novel Fc-Optimized CD133 Antibody

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3793-3793 ◽  
Author(s):  
Kathrin Rothfelder ◽  
Samuel Koerner ◽  
Maya Andre ◽  
Julia Leibold ◽  
Philaretos Kousis ◽  
...  
Keyword(s):  
Nk Cells ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Ex Vivo ◽  
Leukemic Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor

Abstract NK cells largely contribute to the success of monoclonal antibody (mAb) application in cancer due to their ability to mediate antibody-dependent cellular cytotoxicity (ADCC), a feature considered critical for therapeutic success. Up to now, no immunotherapeutic antibodies are available for the treatment of myeloid leukemias. Recently, we reported on the development of mAb targeting CD133, which is expressed on a wide variety of tumor cells (Koerner et al., Blood 2014 124:2309). Here we extend our analyses and provide further data on the preclinical characterization of an Fc-engineered CD133 mAb for the treatment of myeloid leukemia. Compared to two other anti-human CD133 mAb (clones AC133 and W6B3), which both bound to primary AML and CML cells in 15/25 and 7/10 cases, respectively, clone 293C3 recognized the leukemic cells in 22/25 AML cases and 7/10 CML cases. Based on these results, clone 293C3 was chosen to generate chimeric mAb with either a wildtype Fc part (293C3-WT) or a variant containing amino acid exchanges (S239D/I332E) to enhance affinity to the activating Fc receptor CD16 on NK cells (293C3-SDIE). Treatment with 293C3-SDIE resulted in significantly enhanced activation, degranulation and lysis of primary CD133-positive AML cells by NK cells in allogeneic and autologous experimental ex vivo settings as compared to its wildtype counterpart. Considering the expression of CD133 on healthy hematopoietic progenitor cells, we further performed colony forming unit assays with healthy bone marrow (BM) cells. In line with the observed lower expression levels of CD133 on healthy compared to malignant hematopoietic cells no relevant toxicity of 293C3-SDIE at the level of committed hematopoietic progenitor cells was observed. Moreover, 293C3-SDIE did not induce lysis of of healthy BM cells by allogeneic or autologous NK cells. In a NOD.Cg-Prkdcscid IL2rgtmWjl/Sz (NSG) xenotransplantation model, induction of ADCC by treatment with 293C3-SDIE resulted in the elimination of patient AML cells by NK cells from a matched human donor. Thus, 293C3-SDIE constitutes an attractive immunotherapeutic compound, in particular for the elimination of minimal residual disease in CD133 bearing leukemia in the context of allogenic SCT. Disclosures No relevant conflicts of interest to declare.

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