Suppression of Mir-708 Promotes DKK3 to Inhibit Wnt/β-Catenin Signaling Pathway in Adult B-ALL

Abstract Dickkopf-3 (DKK-3) is a Wnt signaling antagonist, and DKK3 inactivation is associated with poor prognosis in various solid tumors and hematologic malignancies. Epigenetic hypermethylation has been implicated in downregulation of DKK3, but other regulatory mechanisms remain to be investigated. In this study, we examined DKK3 expression and the role of microRNA in the regulation of DKK3 in adult B-ALL. Our results showed low levels of DKK3 expression in Nalm-6 and BALL-1 cell lines, and leukemia cells from adult B-ALL patients. DKK3 expression was remarkably lower in the initial diagnosis and relapse samples than in the matched samples during remission. In addition to hypermethylation, low levels of DKK3 were associated with high expression of miR-708. This finding was similar to that in childhood ALL. The miR-708 was predicted to bind to the 3'-UTR of DKK3. By using miR-708 mimics, we found that miR-708 targets DKK3 to promote B-ALL cell proliferation through cell cycle promotion and apoptosis inhibition. The treatment with 5-aza-2'-deoxycytidine significantly increased DKK3 and decreased p-GSK3β, cyclin D1 and nuclear and cytoplasmic β-catenin protein expression. A synergistic effect was seen when the miR-708 inhibitor and 5-aza were used simultaneously. Although miR-708 was reported to either promote or suppress tumorigenecity in some solid tumors, our findings indicate that suppression of miR-708 increases DKK3 expression to inhibit the Wnt/β- catenin signaling pathway in B-ALL. Disclosures No relevant conflicts of interest to declare.

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Function of Growth Factor Independence 1 (GFI1) in the Polarization AML -Associated Stroma

Blood ◽

10.1182/blood.v124.21.4366.4366 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4366-4366

Author(s):

Lacramioara Botezatu ◽

Yahya S. A. Al-Matary ◽

Bertram Opalka ◽

Jan Dürig ◽

Thomas Schroeder ◽

...

Keyword(s):

Bone Marrow ◽

Solid Tumors ◽

Tumor Cells ◽

Conflicts Of Interest ◽

Leukemic Cells ◽

Leukemia Cells ◽

Human Leukemia ◽

Deficient Mice

Abstract The growth of various solid tumors, lymphoma and leukemias is not only the result of cell-specific changes at the genetic and epigenetic level, but is also affected by the surrounding stroma and the cells therein. It has been shown that tumor cells induce surrounding immune cells to express various cytokines and other factors, which promote further growth and spread of tumor cells. Most studies have been conducted with solid tumors, however, not much is known about the role of stroma cells with regard to haematopoietic tumors. In addition, our knowledge is limited with regard to the cell intrinsic factors governing the polarization of stroma cells. We intended to study the role of stroma cells in acute myeloid leukemia (AML), a malignant disease of the myeloid lineage and in myelodysplastic syndrome (MDS), a disease which is characterized by disturbed function of the bone marrow, which can often progress to AML. We focused our studies on two different components of the stroma: mesenchymal stroma cells (MSC) and monocytes. Bone marrow derived MSCs and monocytes originating from AML patients better supported the growth of human leukemia cells in vitrothan MSCs and monocytes from control healthy persons. Interestingly, after achieving a remission, MSCs and monocytes from the cured patients did not any more support the growth of leukemia cells to the same extent as monocytes and MSCs from leukemic patients. To better understand the mechanism behind this observation, we used different murine MDS and AML models. Similar to the finding in human patients, bone marrow derived MSCs and monocytes from MDS and AML mice better supported the growth of leukemic cells in vitro than monocytes and MDSc from healthy mice. In addition, we observed a higher number of non-malignant MSCs and monocytes in the bone marrow of leukemic mice than in the bone marrow of healthy mice. Furthermore, the higher the number of monocytes was in the bone marrow of the mice, the more aggressive was the course of the leukemia. On a molecular level we found that the transcription factor Gfi1 is 3-4 fold upregulated in the MSCs and monocytes of leukemic mice. To verify whether Gfi1 is indeed required for the polarization of MSCs and monocytes we used leukemic Gfi1-deficient mice. In these mice the accumulation of monocytes was less pronounced than in the bone marrow of Gfi1 wildtype mice. In addition MSCs and monocytes from Gfi1-deficient mice did not support to the same extent the growth of leukemic cell lines in vitro, as did the monocytes and MSCs of Gfi1 wildtype leukemic mice. Thus, we have first indications that MSCs and monocytes play an important role in the support of leukemic cells and that Gfi1 is involved in the polarization of these cells and thus Gfi1 could serve as an additional approach to treat leukemia and MDS. Disclosures No relevant conflicts of interest to declare.

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Role of transforming growth factor-β in hematologic malignancies

Blood ◽

10.1182/blood-2005-10-4169 ◽

2006 ◽

Vol 107 (12) ◽

pp. 4589-4596 ◽

Cited By ~ 154

Author(s):

Mei Dong ◽

Gerard C. Blobe

Keyword(s):

Growth Factor ◽

Signaling Pathway ◽

Transforming Growth Factor ◽

Myeloproliferative Disorders ◽

Transforming Growth Factor Β ◽

Hematologic Malignancies ◽

Negative Regulator ◽

Cellular Processes ◽

Signaling Field

AbstractThe transforming growth factor-β (TGF-β) signaling pathway is an essential regulator of cellular processes, including proliferation, differentiation, migration, and cell survival. During hematopoiesis, the TGF-β signaling pathway is a potent negative regulator of proliferation while stimulating differentiation and apoptosis when appropriate. In hematologic malignancies, including leukemias, myeloproliferative disorders, lymphomas, and multiple myeloma, resistance to these homeostatic effects of TGF-β develops. Mechanisms for this resistance include mutation or deletion of members of the TGF-β signaling pathway and disruption of the pathway by oncoproteins. These alterations define a tumor suppressor role for the TGF-β pathway in human hematologic malignancies. On the other hand, elevated levels of TGF-β can promote myelofibrosis and the pathogenesis of some hematologic malignancies through their effects on the stroma and immune system. Advances in the TGF-β signaling field should enable targeting of the TGF-β signaling pathway for the treatment of hematologic malignancies.

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The Role of Beta 2 Integrins in Macrophage Migration During Resolution of Inflammation.

Blood ◽

10.1182/blood.v114.22.3600.3600 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3600-3600 ◽

Cited By ~ 1

Author(s):

Yevgeniya Kushchayeva ◽

Darya Mishchuk ◽

Tatiana Ugarova

Keyword(s):

Lymph Nodes ◽

Conflicts Of Interest ◽

Fold Increase ◽

Initial Population ◽

Blood Monocytes ◽

Resident Cell ◽

Low Levels ◽

Inflammatory Macrophages ◽

Β2 Integrins

Abstract Abstract 3600 Poster Board III-537 The mobilization of blood monocytes and their differentiation into macrophages during the immune-inflammatory response helps to prepare the tissue for resolution. During the resolution phase of inflammation macrophages do not die locally: some cells emigrate by draining lymphatics whereas some remain at the site of inflammation. The major myelo-monocytic integrin αMβ2 (Mac-1, CD11b/CD18), together with two related integrins αDβ2 (CD11d/CD18) and αXβ2 (CD11c/CD18), mediate critical adhesive reactions of monocyte/macrophages. However, the roles of these adhesion receptors in control of macrophage retention at sites of inflammation and their emigration to lymph nodes are unclear. Using a mouse model of sterile peritonitis induced by thioglycollate injection, we examined the dynamics of macrophage β2 integrins during the resolution phase of inflammation. Macrophages were defined by FACS analyses as a population of cells expressing αMβ2high, αDβ2+ and CD115+. The initial population of resident β2, positive for βDβ2 and negative for αXβ2. The thioglycollate-challenged mice showed a ∼4-fold increase in macrophages on day 3 followed by a progressive decrease to normal resident cell numbers by day 13. Expression of αMβ2 on macrophages on day 3 decreased by 2.5-fold as a result of dilution of the initial population of αMβ2high resident macrophages by infiltrating blood monocytes expressing αMβ2low. However, after day 3, the density of αMβ2 on macrophages gradually increased and by day 13 returned to the high levels characteristic of resident macrophages. By contrast, expression of αDβ2 and αXβ2 on inflammatory macrophages increased by 2-fold by day 6-9 compared to that on resident macrophages and then returned to the resident levels by day 3. Thus, although the number of macrophages decreased from day 3 to day 9 by several fold, the population of macrophages which remained in the peritoneum was enriched in cells expressing the high levels of αMβ2 and αDα2. Tracking migration of fluorescently labeled peritoneal cells demonstrated that a population of macrophages which leaves the inflamed peritoneum and enters lymph nodes consists of cells expressing low levels of αMβ2 and αDβ2. These data suggested that upregulation of β2 integrins, especially αMβ2, may be responsible for the retention of macrophages in the peritoneum. Indeed, the rate of macrophage emigration from the peritoneum in the αMβ2-deficient mice was significantly higher than that in wild-type mice. The results indicate that macrophage emigration from the inflamed site is controlled by the level of integrin αMβ2 and αDβ2 with low expressors being migratory and high expressors remaining in the peritoneum. The data also highlight the importance of integrins αDβ2 and αXβ2 as specific markers of inflammatory macrophages. Disclosures: No relevant conflicts of interest to declare.

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Impaired Hydroxylation of 5-Methylcytosine In TET2 mutated Patients with Myeloid Malignancies

Blood ◽

10.1182/blood.v116.21.1.1 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1-1

Author(s):

Anna Jankowska ◽

Myunggon Ko ◽

Yun Huang (equal contribution) ◽

Utz J. Pape ◽

Hadrian Szpurka ◽

...

Keyword(s):

Mrna Expression ◽

Genomic Dna ◽

Conflicts Of Interest ◽

Methylation Pattern ◽

Myeloid Malignancies ◽

Cpg Sites ◽

Low Levels ◽

Methylation Patterns ◽

The Impact

Abstract Abstract 1 TET2 mutations are frequently found across broad spectrum of myeloid malignancies but how these mutations contribute to diseases is still unknown. Preliminary results from our laboratory have suggested that TET2 converts 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) and consequently, the levels of 5-hmC may be lower in genomes of mutant bone marrow cells. To facilitate study of TET2 function we developed a blot assay to detect 5-hmC in genomic DNA with a specific antiserum to 5-hmC. In a second improved assay with increased sensitivity and precision, we treated genomic DNA with bisulfite in order to convert 5-hmC to cytosine 5-methylenesulfonate (CMS) and measured 5-hmC levels indirectly using a specific anti-CMS serum. Based on the results of this technique we demonstrate here for the first time that indeed TET2 mutations in predicted catalytic residues and other positions compromised TET2 function. We studied 102 patients with various myeloid malignancies (4/28 MDS, 14%, 26/48 MDS/MPN, 54% and 1/4 MPN, 2% and primary 2/11 AML 18% and 3/11 sAML, 27% TET2 mutants, respectively) and compared to wt cases or controls (N=17). Mutations were found throughout the entire coding region and were mostly inactivating (33/45 TET2 mutations). The levels of 5-hmC in genomic DNA from TET2 mutants were significantly decreased in comparison to wt cases and controls (p=4.5e-08 and p=1.8e-09, respectively). Particularly low levels of 5-hmC were found in patients with homozygous (UPD)/hemizygous (deletion) TET2 mutations and those with biallelic mutations. Surprisingly, 18% of all TET2 WT patients also showed low levels of genomic 5-hmC (despite normal TET2 mRNA expression), suggesting that these patients may carry not yet identified variants/lesions in TET2 or other partner proteins involved in TET2-mediated catalysis. To further investigate the impact of TET2 mutations associated with myeloid malignancies we also introduced 9 different missense mutations corresponding to those found in patients into murine Tet2 cells; severe loss of enzymatic activity was observed in 7/9 cases as measured by greatly diminished 5-hmC levels. To study the role of Tet2 in normal hematopoiesis we depleted Tet2 in C57BL/6 mice by retrovirus-mediated transduction of shRNA against Tet2. Tet2 depletion is associated with skewing of hematopoietic differentiation towards the monocyte/macrophage lineage. To further investigate the function of TET2 we transduced the myeloid THP-1 cell line with lentiviral vector containing TET2 cDNA (TET2+) or an empty vector. This manipulation allowed us to select clones showing 19-fold increase in TET2 mRNA expression without significantly alterations of proliferation kinetics. Using this model we studied the impact of TET2 overexpression on resultant methylation pattern of CpG sites. We have applied Illumina Infinium HumanMethylation27 arrays (27,5K CpG sites/14.4K genes). Overexpression of TET2 resulted in a distinct promoter methylation patterns with 169 altered CpG sites with difference of averaged β>0.5 (considered significant as compared to control). Among these differentially methylated loci, 27 promoters were significantly hypomethylated while 42 were hypermethylated as compared to control cells. Change in methylation pattern observed through overexpression of TET2 in vitro prompted us to analyze methylation patterns in patients with and without TET2 mutations or those with decreased 5-hmC levels. Using methylation arrays a total of 62 cases were analyzed. When patients were grouped based on the levels of 5hmC, an associated methylation signature can be clearly discerned with 2512 differentially methylated loci and distinct skewing towards hypomethylation (2510 sites; e.g., TMEM102, ABCC11) vs. hypermethylation (2 sites, AIM2 and SP140), consistent with the observation made in the TET2+ cells line. In sum, our results provide strong evidence for TET2 as the first mutated gene in myeloid malignancies that is involved in conversion of 5-mC to 5-hmC in DNA, indicating the novel role of TET2 in a substantial component of epigenetic deregulation in myeloid malignancies. Disclosures: No relevant conflicts of interest to declare.

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Re-Expression of the AML1/ETO Target Gene LAT2/NTAL/LAB Results In Direct Interference with Myeloid Differentiation In AML1/ETO-Positive Cells

Blood ◽

10.1182/blood.v116.21.2474.2474 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2474-2474

Author(s):

Jesus Duque-Afonso ◽

Aitomi Essig ◽

Leticia M Solari ◽

Tobias Berg ◽

Heike L. Pahl ◽

...

Keyword(s):

Western Blot ◽

Cell Lines ◽

Functional Role ◽

Target Gene ◽

Conflicts Of Interest ◽

Myeloid Cell ◽

Leukemia Cells ◽

Myeloid Differentiation ◽

Granulocytic Differentiation

Abstract Abstract 2474 Background: The leukemia-specific oncofusion protein AML1/ETO regulates different target genes, including the LAT2 gene (encoding the adaptor molecule LAT2/NTAL/LAB), which is epigenetically repressed by AML1/ETO as we have previously described. LAT2 is phosphorylated by c-kit and has a role in mast cell and B cell activation. To address the functional role of LAT2 during myeloid differentiation, expression studies were performed in myeloid cell lines, and LAT2 was overexpressed by retroviral gene transfer in AML1/ETO-positive Kasumi-1 cells and AML1/ETO-negative U-937 cells. Methods: To induce monocytic and granulocytic differentiation, the myeloid cell lines U-937, HL-60 and NB4 were treated with PMA and ATRA, respectively, and LAT2 expression measured by both Northern and Western blot. LAT2 was overexpressed in Kasumi-1 and U-937 cells by use of the retroviral vector pMYSiG-IRES-GFP. Virus was produced in 293T cells and titrated in TE671 cells. Following transduction, GFP-positive cells were sorted by fluorescence-activated cell sorting (FACS). Transduced cells were treated with PMA (2 and 10 nM for 24 and 48 hours) and ATRA (0.1 μM and 0.5 μM for 48 and 96 hours), respectively. Results: The AML1/ETO-negative myeloid cell lines HL-60, NB4 and U-937 readily expressed LAT2, which was further upregulated by PMA, and transiently downregulated with ATRA. In the AML1/ETO-positive Kasumi-1 and SKNO-1 cells, LAT2 expression was absent. To address the functional role of this repression, forced expression of LAT2 was achieved in Kasumi-1 and U-937 cells and resulted in effective processing of LAT2 protein (confirmed by Western blot), and a decrease in the expression of the differentiation markers CD11b and CD11c (FACS analysis) in Kasumi-1 but not U-937, with only minor effects of LAT2 overexpression upon apoptosis and cell growth arrest. Notably, during both PMA- and ATRA-induced differentiation, a striking maturation block occurred in Kasumi-1 (measured by CD11b/CD11c expression, observed at different doses and time points of these treatments), while differentiation of U-937 cells was unimpaired by overexpression of LAT2. Conclusions: In AML1/ETO-negative leukemia cells, LAT2 expression is differentially regulated during monocytic and granulocytic differentiation. In AML1/ETO-positive leukemia cells, in which LAT2 is repressed, LAT2 re-expression imposes a striking maturation block. Graded expression of this novel AML1/ETO target gene may therefore play an important role in maintaining the phenotypic characteristics of this leukemia subtype. Disclosures: No relevant conflicts of interest to declare.

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Flt3/ITD Blocks Myeloid Differentiation Of Hematopoietic Cells By Up-Regulating Runx1

Blood ◽

10.1182/blood.v122.21.3808.3808 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3808-3808

Author(s):

Tomohiro Hirade ◽

Mariko Abe ◽

Chie Onishi ◽

Seiji Yamaguchi ◽

Seiji Fukuda

Keyword(s):

Conflicts Of Interest ◽

Hematopoietic Cells ◽

Cell Number ◽

Dominant Negative ◽

Leukemia Cells ◽

Myeloid Differentiation ◽

Differentiation And Proliferation ◽

Runx1 Expression ◽

Novel Therapeutic

Abstract Internal-Tandem-Duplication mutations in the FLT3 (FLT3/ITD) gene are detected in 30% of patients with acute myeloid leukemia (AML) and are associated with extremely poor prognoses. The lack of significant efficacy of FLT3/ITD inhibitors underscores the need to identify FLT3/ITD-specific signaling pathways that are distinct from those that occur in normal hematopoietic cells to develop novel therapeutic approaches. FLT3/ITD is classified as a “class I mutation” that drives the proliferation of leukemia cells. In addition to mutation of FLT3/ITD, a “class II mutation” that blocks differentiation of the pre-leukemic clone is generally required for the development of AML. For instance, dominant negative mutations of RUNX1 are occasionally found in patients with AML. These mutations of RUNX1 cause AML by blocking the differentiation of leukemia cells in combination with the mutation of FLT3/ITD. RUNX1 is a core-binding transcription factor and plays an important role in hematopoietic homeostasis, particularly differentiation and proliferation. Loss of RUNX1 blocks hematopoietic differentiation and is associated with the emergence of a primitive hematopoietic compartment, suggesting that RUNX1 generally induces differentiation of hematopoietic cells. However, the functional role of RUNX1 as a down-stream effector of FLT3/ITD has not been characterized. Herein, we investigated the role of Runx1 in aberrant proliferation and differentiation of hematopoietic cells induced by Flt3 /ITD. A comparison of RUNX1 expression levels in AML patients for whom information has been deposited in the public gene expression profile database (GSE1159) revealed that RUNX1 mRNA expression was significantly higher in FLT3/ITD+AML cells (N=78) than in FLT3/ITD-AML cells (N=190, P<0.05). The mRNA microarray analysis consistently demonstrated that Runx1 is up-regulated by Flt3/ITD in Ba/F3 cells. Up-regulation of Runx1 by Flt3/ITD was validated in Ba/F3 cells and 32D cells by quantitative RT-PCR. Incubation of control 32D cells with 20 ng/ml of G-CSF increased the number of Gr-1+/Mac-1+cells, whereas the induction of myeloid differentiation by G-CSF was abrogated by the overexpression of Flt3/ITD in 32D cells. By contrast, transduction of shRNA specific for Runx1 into Flt3/ITD+32D cells inhibited the expression of Runx1 mRNA by 60 % but increased the number and the proportion of Gr-1+/Mac-1+cells ; these effects were enhanced by incubation with G-CSF. These data indicate that Runx1 mediates the block of differentiation toward the myeloid lineage that is induced by Flt3/ITD. Moreover, the number of colony-forming units (CFU) over-expressing Flt3/ITD cultured in the absence of growth factors was reduced by Runx1-shRNA without affecting the total cell number in the suspension culture, as compared to Flt3/ITD+32D cells transduced with control-shRNA. This implies that antagonizing Runx1 facilitates the production of terminally differentiated cells that have lost colony-forming ability, thereby reducing the CFU number without altering the total number of cells. Finally, Runx1-shRNA inhibited the formation of secondary CFU colonies derived from the primary Flt3-ITD-over-expressing CFU colonies. Our results suggest that Flt3/ITD blocks myeloid differentiation of Flt3/ITD+cells by up-regulating Runx1 expression. The blocking of differentiation mediated by Runx1 in Flt3/ITD+cells is in contrast to the cell differentiation-inducing role of Runx1 in normal hematopoiesis, suggesting that the function of Runx1 in Flt3/ITD+cells may be distinct from that in normal cells. The reduction of secondary CFU colonies by Runx1-shRNA suggests that Runx1 may mediate self-renewal of Flt3/ITD+hematopoietic progenitor cells. These findings suggest that antagonizing RUNX1 may represent a novel therapeutic strategy to induce terminal differentiation of FLT3/ITD+AML cells in AML patients, in addition to inhibiting their aberrant proliferation. Disclosures: No relevant conflicts of interest to declare.

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Angiogenic switch during 5T2MM murine myeloma tumorigenesis: role of CD45 heterogeneity

Blood ◽

10.1182/blood-2003-08-2946 ◽

2004 ◽

Vol 103 (8) ◽

pp. 3131-3137 ◽

Cited By ~ 37

Author(s):

Kewal Asosingh ◽

Hendrik De Raeve ◽

Eline Menu ◽

Ivan Van Riet ◽

Eric Van Marck ◽

...

Keyword(s):

Disease Progression ◽

Solid Tumors ◽

Flow Cytometric Analysis ◽

Active Role ◽

Hematologic Malignancies ◽

Enzyme Linked Immunosorbent Assay ◽

Angiogenic Switch ◽

Tumor Neovascularization ◽

End Stage

Abstract The active role of angiogenesis during disease progression is well recognized in solid tumors. In hematologic malignancies such as multiple myeloma (MM), it is not known whether tumor neovascularization is an epiphenomenon or whether it is actively involved in disease progression. At clinical presentation, myeloma disease and the associated angiogenesis are both well established. Here the 5T2MM murine model was used to analyze angiogenesis during preclinical myeloma stages. Bone marrow (BM) of 5T2MM-inoculated mice was analyzed at weekly intervals until the end stage of the disease. Histologic analysis and assessment of microvessel density (MVD) by CD31 staining demonstrated a preangiogenic stage of small tumor aggregates followed by an angiogenic switch and subsequently an angiogenic stage of progressive tumor growth and large, confluent tumor nodules. Flow cytometric analysis that indicated an increase in percentage CD45- MM cells preceded the angiogenic switch. Real-time polymerase chain reaction (RT-PCR) of sorted CD45+ and CD45- MM cells indicated higher vascular endothelial growth factor 120 (VEGF120) and VEGF164 transcripts in CD45- MM cells. VEGF enzyme-linked immunosorbent assay (ELISA) revealed high secretion by CD45- MM cells but no protein secretion by CD45+ MM cells, indicating angiogenic heterogeneity among the MM cells. These data suggest that, like in solid tumors, angiogenic switch and angiogenic heterogeneity exist in MM. (Blood. 2004;103:3131-3137)

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Synthesis of eosinophil-associated enzymes in HL-60 promyelocytic leukemia cells

Blood ◽

10.1182/blood.v68.1.185.185 ◽

1986 ◽

Vol 68 (1) ◽

pp. 185-192 ◽

Cited By ~ 14

Author(s):

SA Fischkoff ◽

GE Brown ◽

A Pollak

Keyword(s):

Endoplasmic Reticulum ◽

Protein Synthesis ◽

Promyelocytic Leukemia ◽

Leukemia Cells ◽

Acute Myelomonocytic Leukemia ◽

Granule Morphology ◽

Synthesis And Processing ◽

Low Levels ◽

Myelomonocytic Leukemia

Abstract Eosinophils derived from HL-60 cells share many of the abnormalities of granule histochemistry and morphology frequently seen in eosinophils of patients with certain malignancies, especially those seen in acute myelomonocytic leukemia with abnormal eosinophils (FAB class M4eo). In order to understand the pathogenesis of these abnormalities, four enzymes, characteristic of the eosinophil, were studied in HL-60 promyelocytic leukemia cells at various stages of eosinophilic differentiation. Using biochemical and ultrahistochemical techniques, the following differences from normal eosinophil development were demonstrated. First, both myeloperoxidase and eosinophil peroxidase coexisted in the population of maturing HL-60 eosinophils. Second, the granules formed from the condensation of material in vacuoles which were derived from dilated segments of the endoplasmic reticulum; the role of the Golgi apparatus in processing of peroxidase appeared minimal. Third, low levels of lysophospholipase and arylsulfatase were present in the cells compared to normal eosinophils. Finally, crystallizations resembling precursor structures of Auer rods appeared in the granules of about 5% of the cells. These findings suggest that several disorders of the control of protein synthesis and processing exist in HL-60 eosinophils which may be responsible for the abnormal granule morphology and histochemistry.

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Role of STAT3 in Immunoregulatory Function of Human Bone Marrow-Derived Mesenchymal Stem Cells.

Blood ◽

10.1182/blood.v114.22.3637.3637 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3637-3637

Author(s):

Jinsun Yoon ◽

Seoju Kim ◽

Eun Shil Kim ◽

Byoung Kook Kim ◽

Young Lee

Keyword(s):

T Cells ◽

Conflicts Of Interest ◽

Hematologic Malignancies ◽

Treg Cells ◽

Human Mscs ◽

Hematopoietic Stem ◽

The One

Abstract Abstract 3637 Poster Board III-573 The one of the best curative treatment modality in hematologic malignancies is an allogeneic hematopoietic stem cell transplantation (HSCT). However, graft-versus-host disease (GVHD) is a major obstacle of allogeneic HSCT. BM derived human MSCs are known to have immunoregulatory effect in vitro and in vivo via inhibiting alloreactive T lymphocytes, leading to their clinical use for the prevention of GVHD in HSCT. However, the molecular mechanism of immunoregulatory effect of human MSCs is not fully understood. In this study, the signaling of immunoregulatory effect was investigated by co-culture of human MSCs with lymphocytes. The proliferation of allogeneic T cells was inhibited by MSCs. Among the STATs, STAT3 was a key molecule in MLR co-cultured with MSCs. STAT3 siRNA treated MSCs did not inhibit the lymphocyte proliferation. After MSCs were trasnsfected with STAT3 plasmid, the fraction of CD4+CD25+FOXP3+ cells (Treg cells) were increased, while the fraction of CD4+, CD8+, CD25+ was decreased. In addition, Th1-related cytokines (IL-2, IL-12 and INF-γ) and Th17-related cytokines (IL-6, IL-17 and IL-21) were down-regulated, and Th2-related cytokines (GATA-3, IL-4 and IL-10) were up-regulated in MLR co-cultured with STAT3-ablated MSCs, while vice versa in MLR co-cultured with STAT3-transfected MSCs. Furthermore, ELISA showed that concentration of Th1-related cytokine (IL-2) in the supernatant of MLR co-cultured with STAT3-ablated MSCs was higher than that of control; while concentration of Th2-related cytokine (IL-4) was lower than that of control. These results suggested that induction of Th1 to Th2 shift by MSCs might be mediated via STAT3 molecule. In summary, STAT3 may be an indispensable molecule in the immunoregulatory effect in human MSCs via modulation of regulatory T cells. Disclosures: No relevant conflicts of interest to declare.

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