bluuebunny/crossref_metadata_2025 huggingface dataset:
Therapeutic Potential of Ovarian Monocyte Progenitor Cells (MPCs) in SLE-Murine Model.
Authors: El-Badri (Dajani) Nagwa S. , Cajera Vaishali , Hakki Amal , Baldwin Margaret , Sanberg Paul R.
Published: April 2020

We have previously shown that the progeny of resident ovarian stromal progenitor cells is restricted to the production of cells of the monocyte-macrophage lineage. These ovarian monocyte progenitor cells (MPCs) formed hematopoietic colonies on methyl cellulose, and were CD45+, CD11b+, CD11c+, and Ly6-Gr-1. A distinct CD45low population within these cells reacted with CD117 (C-kit) surface marker, suggestive of a primitive hematopoietic progenitor. To determine the therapeutic potential of MPCs, we transplanted 50,000 cells into normal and autoimmune BXSB autoimmune mouse model for SLE. MPCs did not rescue lethally irradiated mice from the consequences of irradiation, and were not engrafted in MHC-mismatched mouse strains. However, murine SLE-like symptoms were improved, and substantial reduction in inflammatory infiltrate was observed in tissue sections from transplanted mice including kidneys, lungs, and spleen. Restructuring of renal glomerural sclerosis was also observed in the transplant recipients. To determine whether glomerular vascular regeneration was directly mediated by donor MPCs, we tested the hypothesis that MPCs co-express markers for endothelial progenitor cells and monocytes. After culture in conditions permissible to endothelial cell differentiation, large percentage of MPCs (73%) expressed endothelial cell marker CD106. When their culture supernatant was applied to a rat aortic ring in matrigel tube forming assay, robust tube formation was observed in the experimental group. However, similar tube formation was also observed in the control well containing 20% FBS, and cultured MPCs failed to produce tube formation in matrigel assay. Interestingly, phenotypic analysis showed that only 52% of MPCs express MHC class I suggesting that these cells may induce less rejection when transplanted in mismatched hosts. These data suggest that vascular tissue repair initiated by MPCs is mediated by anti-inflammatory factors, and not by direct contribution to the reconstruction of glomeular vasculature.
Microarray Analysis of Thrombopoietin Induced Lineage Commitment of Bi-Potential Cell Line HU-3.
Authors: Morgan Doris A. , Jost Monika , Bender Patrick , Mayo Kelly , Kriete Andres
Published: April 2020

HU-3 is a cytokine dependent cell line derived from a patient with megakaryoblastic leukemia. The cells have

a strong megakaryocytic phenotype when cultured in GM-CSF and undergo terminal erythropoiesis when switched to stem cell factor and erythropoietin. (Morgan, 1997).

HU-3 cells are equally responsive to thrombopoietin (TPO), but no longer can be induced into the erythroid pathway nor do they advance along the megakaryocyte differentiation pathway. Microarray analysis of the bi-potential and unipotential cells using arrays with genome-wide coverage and bioinformatics software identified a panel of genes associated with the loss of erythroid potentiality and with an apparent commitment to the megakaryocytic lineage. Down-regulation of erythroid-specific genes expressed in the bi-potential cells was the predominant effect of the TPO microenvironment.

Expression Readouts of Globin and Other Erythroid Genes HU-3 alpha globin beta globin gamma globin zeta globin epsilon globin Rh factor GlycophorinA GM 187 32 258 17 94 46 80 TPO 50 4 130 2 9 4 2

HU-3 (TPO) did not show an enhanced megakaryocyte phenotype nor increased expression of CD41 (GpIIb), CD42 (Gp Ib) CD61 (GpIIIa), CD62 (P-selectin); Gp V, Gp IX, VEGF, and PDGF; however, several megakaryocyte-linked genes were up-regulated: pro-collagen lysine, annexin A1, angiopoietin 2, beta 1 integrin, and galactin 1 among others. Interleukin 8 expression increased approximately 30-fold in the TPO cultures. AML1 (RUNX1), GATA-1, and SPi-1/PU.1 are reported to be required for megakaryocyte and erythroid maturation, but were expressed at low levels and were not up-regulated by TPO. This observation may explain the block in the ability of HU-3 to enter the maturation pathway once unipotentiality had been achieved. The HU-3 cell line is a biologically relevant in vitro system for exploring the genetic program at the onset of megakaryocyte commitment and identifying genes necessary for entry into the maturation pathway.
N-Acetyl Cysteine Reduces von Willebrand Factor Multimer Size In Vitro and In Vivo.
Authors: Chen Junmei , Gushiken Francisca C. , Nolasco Leticia , Moake Joel F. , Lopez Jose A.
Published: April 2020

von Willebrand factor (VWF) shares a similar domain structure with many polymeric mucins, including the presence of D domains and a C-terminal cysteine knot, which allows these molecules to form polymeric structures that can reach immense sizes. Precipitation of polymeric lung mucins complicates the clinical course of cystic fibrosis and chronic obstructive lung disease, and in both cases the viscosity of the inspissated mucus can be reduced by treatment with N-acetyl cysteine (NAC), which presumably reduces the size of the mucin polymers by reducing sensitive disulfide bonds. Because of the similarity of VWF and mucin multimers, we examined whether NAC could also reduce VWF multimer size both in vitro and in vivo. In vitro, we incubated NAC at different concentrations and for different times with ultra-large VWF multimers (ULVWF) isolated from endothelial cell supernatant and examined the effect on multimer size using agarose gel electrophoresis. NAC reduced ULVWF size in a time- and concentration-dependent manner, with the peak effect reached at 5 min and at concentration of 0.5 mM. We then examined the effect of NAC on ULVWF/platelet “strings” formed on the surface of histamine-activated endothelial cells by perfusing the strings with NAC solutions. At 1 mM, NAC eliminated almost all of the adherent strings within 5 minutes. We next examined the effect of NAC in vivo by following VWF multimer size with time in C57B/6 injected with NAC either intraperitoneally or intravenously. NAC, at a single dose of 500 mg/kg, induced a sustained reduction in VWF multimer size in the treated mice within 4 hours after injection. The effects lasted up to 8 hours. These results suggest that NAC may be a rapid, safe, and effective treatment for patients suspected of suffering from thrombotic thrombocytopenic purpura, a disorder characterized by a failure to process ULVWF.
Alternation of Cellular Morphology and Proliferation Induced by microRNA in Human Leukemia Cell Line, UT-7/EPO.
Authors: Kosaka Nobuyoshi , Yamamoto Yusuke , Nogawa Nami , Sugiura Keiichi , Miyazaki Hiroshi , Komatsu Norio , Ochiya Takahiro , Kato Takashi
Published: April 2020

Mature microRNA (miRNA) originated from primary miRNA (pri-miRNA) is a new group of potential regulator for cell differentiation, apoptosis, proliferation and oncogenesis. Some miRNAs were recently identified in hematopoietic cells, while the roles of miRNAs in erythrocytic and megakaryocytic cells had not been well examined. As a first step to explore for miRNAs specific for hematopoietic lineage, the expressions of several known primary microRNAs in erythrocytic and megakaryocytic cell lines, such as TF-1, HL-60, HEK293 and UT-7 leukemia cells, were examined by RT-PCR. We consequently focused on the pri-miR-10a, a primary transcript of miR-10a located within Hox gene clusters, and found the significant expression in TF-1 cells and UT-7/EPO cells. The UT-7/EPO cells were a subline established from the original UT-7 cells, as well as UT-7/GM and UT-7/TPO cells; therefore it was suitable for the further comparative analysis. Interestingly, in UT-7/EPO cells, the expression of pri-miR-10a increased under stimulation of erythropoietin (EPO; 1U/mL and 10U/mL). Based on these observations, it was postulated that pri-miR-10a might involve in modulating erythrocyte differentiation or proliferation. To clarify the role of pri-miR-10a in UT-7/EPO, we have established clonal cell lines by transfecting UT-7/EPO cells with either the control vector or the pri-miR-10a expression vector pCMV-pri-miR10a. Overexpression of pri-miR-10a in the UT-7/EPO cell line (miR10a-UT-7/EPO) was confirmed by RT-PCR. MiR10a-UT-7/EPO showed higher proliferation rate even at low concentration of EPO (0.1 mU/mL). Overexpression of pri-miR-10a did not appear to affect HOXB4 and HOXA1 expression, as similar mRNA levels were seen in both cell lines. It was notable that the cellular size of miR10a-UT-7/EPO became larger than its parental cells. Morphological studies of miR10a-UT-7/EPO were performed in detail. It is possible that miR-10a was capable to modulate morphological features particularly in cellular size relating to cell cycle regulation. For instance, loss of the E2F family members result in marked macrocytic anemia with megaloblastic features in adult mice (Mol Cell. 2000 Aug;6(2):281–91., Mol Cell Biol. 2003 May;23(10):3607–22., Blood. 2006 Aug 1;108(3):886–95.). Data presented here hypothesized that the roles of miR-10a in erythroid cells are tightly associated with cell cycle.
Novel Role for EKLF in Megakaryocyte-Erythroid Differential Lineage Commitment.
Authors: Frontelo Maria Pilar , Manwani Deepa , Galdass Mariann , Karsunky Holger , Gallagher Patrick G. , Bieker James J.
Published: April 2020

Megakaryocytes and erythroid cells are thought to derive from a common progenitor. Although a number of transcriptional regulators are important for this process, they do not explain the bipotential result. We have used gain- and loss-of-function studies, expression profiling, and molecular analyses to show that EKLF, a transcription factor whose crucial role in erythroid gene regulation is well established, plays an unexpected function in the lineage decision between megakaryopoiesis and erythropoiesis. It achieves this by inhibiting the formation of megakaryocytes derived from the common megakaryocyte-erythroid precursor (MEP) cell, while at the same time stimulating erythroid differentiation. Quantitative examination of EKLF expression during hematopoiesis reveals that, unlike genes whose presence is required for establishment of both lineages, EKLF expression is uniquely down-regulated in megakaryocytes after formation of the MEP. Microarray and molecular analyses support these observations and suggest that megakaryocytic inhibition is achieved, at least in part, by EKLF repression of Fli-1 message levels. These results reveal for the first time that EKLF plays a directive role in erythroid and megakaryocyte lineage decisions prior to establishment of the red cell compartment and suggest a model for transcription factor antagonism in MEP differential lineage commitment. Tests as predicted by this model will be discussed.
PU.1 Activity Determines Fate of Myeloid Progenitor Cells during Lineage Commitment.
Authors: Pospisil Vitek , Necas Emanuel , Stopka Tomas
Published: April 2020

Myeloid cell commitment is regulated by factors interacting with chromatin in a progenitor cell entering differentiation. PU.1 is an ETS family transcription factor that has been well characterized in inducing myelopoiesis and blocking erythroid differentiation. Conditionally activated PU.1-Estrogen Receptor transgene in mouse PU.1 knockout-derived hematopoietic progenitors is known to induce macrophage differentiation. We observed that manipulation of PU.1 activity by using different levels of PU.1-ER activator, tamoxifen, was capable of producing major populations of myeloid progeny including macrophages, granulocytes, lymphocytes and mastocytes. Gradual activation of PU.1 produce distinct committed populations that require presence of PU.1 activator. Withdrawal of tamoxifen from macrophages restored cycling and pluripotent potential resembling parental cell line. Furthermore, very low PU.1 activity induced frequent chromosomal aberations such as polyploidy. To determine that PU.1 directly caused these phenotypic changes we determined its occupancy at genes representing PU.1 targets by quantitative chromatin immunoprecipitation (ChIP). We demonstrate that PU.1 is enriched at regulatory regions of lineage restricted cell populations including promoters of CD11b, CD14, IL7R, PAX5, RAG1, Gelatinase, Lysozyme, and MCP. Different PU.1 levels promote distinct cell populations that have different localizations of PU.1 in chromatin. We also tested mRNA levels of PU.1 target genes in progenitor cell line induced by PU.1. Macrophage-specific target genes such as CD11b and CD14 are induced at high PU.1 levels and granulocytic PU.1 targets such as Gelatinase and Lysozyme are induced at intermediate and high PU.1 levels. B-cell specific mRNAs such as IL7-R, PAX5, and RAG1 were induced at low and high PU.1 levels. Our data suggest that PU.1 is recruited to and transactivates its lineage-restricted target genes differently at distinct PU.1 levels and such mechanism may be involved in myeloid cell commitment and leukemogenesis.
C-MYB and C/EBP Family Transcription Factors Regulate the Gfi-1 Promoter.
Authors: Dahl Richard , Owens Kristin S.
Published: April 2020

Gfi-1 −/− mice generate abnormal immature myeloid cells exhibiting characteristics of both monocytes and granulocytes. One of Gfi-1’s critical functions is to downregulate monocyte specific genes in order for granulocytes to develop properly. Since the transcription factors C/EBP alpha and C/EBP epsilon are needed for granulocyte development we hypothesized that these factors may regulate Gfi-1 expression. The Gfi-1 promoter contains several putative C/EBP binding sites and we show by electrophoretic mobility shift and chromatin immunoprecipitation that C/EBP family members can bind to some of these sites. However we were unable to see activation of the Gfi-1 promoter by C/EBP proteins in transient transfection reporter assays. Other groups have shown that C/EBP proteins can synergize with the transcription factor c-myb. We observed that the Gfi-1 promoter contains sites for the hematopoietic transcription factor c-myb. Sevral of these c-myb binding sites are adjacent to C/EBP binding sites. In reporter assays in non-hematopoietic cells c-myb activated the Gfi-1 promoter by itself and this activity was enhanced when we included either C/EBP alpha or epsilon in the transfection. Our data suggests that C/EBP proteins and c-myb regulate the transcription of Gfi-1 in myeloid cells.
PBI-1402: A Non-Toxic Immunorestorative Small Molecule for the Treatment of Anemia.
Authors: Lagraoui Mouna , Grouix Brigitte , Julien Nathalie , Asselin Michel , Gaudreau Dannyck , Sarra-Bournet François , Morin Marie-Josée , Krosl Gorazd , Roy Denis-Claude , Penney Christopher , Gagnon Lyne
Published: April 2020

Erythropoiesis is regulated by an intricated network of transcription factors and other molecules that mediate differentiation of stem cell progenitors into erythroid lineage. PBI-1402 is a non-toxic, well-defined chemo- and radio-protective orally active small molecule which stimulates erythropoiesis. In vivo studies demonstrate that PBI-1402 has an immunorestorative effect in anemia induced by phenylhydrazine or lethal irradiation. In phenylhydrazine-induced anemia, PBI-1402 treated mice had an increased number of hematopoietic progenitor cells compared to the control mice. This increase was more pronounced in the erythroid lineage (BFU-E and CFU-E). In myeloablated mice that received a syngeneic bone marrow transplant, oral treatment with PBI-1402 resulted in a significant increase in peripheral erythrocyte count and hemoglobin concentration. In conclusion, these results indicate that PBI-1402 plays an important role in the formation of red blood cells. Therefore, PBI-1402 may be useful for the treatment of anemia associated with chemotherapy, radiotherapy, bone marrow transplantation and cancer.
A New Role for Flavoproteins in the Control of Megakaryocyte Polyploidy.
Authors: Makitalo Maria , McCrann Donald J. , Liu Kenian , Ravid Katya
Published: April 2020

Megakaryocytes (MKs) can reach a ploidy level of greater than 32N (with 2N representing diploid chromosomes), while polyploidization is important for generating an adequate number of functional platelets. The MK population in the marrow intriguingly consists of a large fraction of 8N and 16N cells that are cell cycle arrested. Here, we examined the expression of different NADPH (NOX) oxidases in MKs, using Western blotting and immunohistochemistry, because of their known role in upregulating regulators of the G1 phase of the cell cycle and in promoting cell cycle progression. It was found that in thrombopoietin-treated mouse bone marrow cultures, NOX1 is highly expressed in many, but not all MKs, while NOX4 is expressed at lower levels than NOX1. Treatment of mouse bone marrow or fetal liver cultures with a NOX inhibitor, diphenylene iodonium (DPI) lead to accumulation of viable MKs at diploid and tetraploid levels, and to a significant reduction in the fraction of high ploidy MKs, as measured by Flow cytometry analysis with a MK specific marker as well as by immunostaining. MKs isolated by an albumin gradient were subjected to Western blotting, which indicated a decrease in the level of the G1 phase cyclin, cyclin E, in DPI-treated cells. Taken together, this study shows for the first time the expression of NOX isoforms in MKs and highlights the important role of flavoproteins in promoting polyploidy in this lineage. It is suggested that the population of cell cycle-arrested intermediate-ploidy MKs is prevented from reaching higher ploidy, partly because of diminished levels of G1 phase regulators via NOX downregulation.
The Role of Cyclin C on Hematopoietic Cells.
Authors: Miyata Yasuhiko , Lie Yan , Sashida Goro , Nimer Stephen
Published: April 2020

The regulation of pluripotent hematopoietic stem cells (HSCs) is essential for the maintenance of multilineage blood production throughout life. The ability to self-renew or differentiate is a critical factor in the regulation of HSC numbers and cell fate, but so is the ability to enter and reside in a quiescent state (G0 phase of the cell cycle). The regulation of the G0 stem cell compartment is important for maintaining an inexhaustible pool of HSCs, protected from cellular stress. We have recently shown that the ETS transcription factor MEF is an important regulator of HSC quiescence (H. D. Lacorazza, Cancer Cell, 2006). The mechanism by which MEF or cell cycle regulatory proteins like p21 control entry into (and exit from) the G0 stage of the cell cycle is poorly understood. Insights into the mechanisms of HSC regulation could have important therapeutic potential.

Recently, a Cyclin C/cdk3 interaction was reported to play an important role in cell cycle re-entry from quiescent state (G0 to G1) by phosphorylating the retinoblastoma protein (Rb) in human fibroblasts (S. Ren, Cell, 2004). To elucidate if cyclin C is involved in the regulation of cell cycle in hematopoietic cells, especially human CD34+ HSCs, we have optimized a FG12 lentivirus based shRNA strategy (kindly provided by D. Baltimore, PNAS, 2002). shRNA directed against cyclin C successfully decreased both cyclin C mRNA and protein expression levels. Using this vector, we have consistently achieved a 90 % transduction efficiency and a 60–70 % reduction of cyclin C levels in human CD34+ cells as evidenced by real time PCR. Now we are analyzing the cell cycle and biological effects of cyclin C knockdown and overexpression on hematopoietic cells using both human CD34+ cells and human leukemic cell lines.
Crosstalk between Murine Liver Sinusoidal Endothelial Cells (LSEC) and Hematopoietic Stem Cells during In Vitro Hematopoiesis.
Authors: Cardier Jose E. , Luna Gonzalo
Published: April 2020

Crosstalk between murine liver sinusoidal endothelial cells (LSEC) and hematopoietic stem cells during in vitro hematopoiesis. G. Luna, J. E. Cardier. Laboratorio de Patologia Celular y Molecular, Centro de Medicina Experimental, Instituto Venezolano de Investigaciones Científicas (IVIC). Apartado 21827, Caracas, Venezuela

We have showed that at the liver there is a specific hematopoietic microenvironment, constituted by the liver sinusoid endothelial cells (LSEC), which are capable to support, in vitro and in vivo, not only the proliferation but also the differentiation and survival of hematopoietic stem cells (HSC). The ability of LSEC to support hematopoiesis could be related to specific hematopoietic molecules expressed by these cells. In this study, we investigate the expression of some cell adhesion molecules (VCAM-1, ICAM-1, and the integrin a4b1 ), and early acting hematopoietic cytokines (IL-6 and GM-CSF), on LSEC cocultured with HSC. The expression of VCAM-1, ICAM-1, α4β1, IL6 and GM-SCF was increased on LSEC cocultured with HSC. In contrast, a significant decrease in the expression of IL-6 and GM-CSF in the HSC derived from the same cocultures was observed. The blockade of VCAM-1 on LSEC reduced significantly the adhesion of HSC to LSEC monolayers, suggesting that this molecule is involved in the binding of HSC to LSEC microenvironments. There were not changes in the expression of the molecules evaluated when the LSEC and HSC were co-cultured in non-contact conditions, suggesting that soluble factors do not participate in regulating the expression of these molecules. Our data shows that during in vitro hematopoiesis, LSEC are activated to express molecules associated with the hematopoiesis process. LSEC activation is regulated by the contact between these cells and HSC. By expressing critical hematopoietic microenvironment molecules, LSEC may regulate the proliferation and differentiation of HSC, during liver extramedullary hematopoiesis.
Myeloblasts Convert into Fibroblastoid Stromal Cells To Create a Microenvironment for Proliferation of Leukemia Blasts in Acute and Chronic Myelogenous Leukemia Cases.
Authors: Tashiro Haruko , Noguchi Mitsuho , Shirasaki Ryosuke , Gotoh Moritaka , Kawasugi Kazuo , Shirafuji Naoki
Published: April 2020

Object: Cancer stem cell has been analyzed in leukemia, which behaves similarly to normal stem cells on their self-renewal and self-conversion into abnormally differentiated cells to make an exact recapitulation of the original heterogeneous leukemia cells. We observed a primary in vitro culture of non-adherent leukemia blasts prepared from various kinds of acute leukemia and chronic myelogenous leukemia cases, and biological and biochemical characteristics were analyzed.

Method: Leukemia blast-rich fractions were prepared from patients’ blood or bone marrow after gradient sedimentation method, which were cultured for a long term. When the appearance of the cultured cells converted into fibroblastoid cells, cells were divided into clones, analyzed molecularly to identify whether they were originated from leukemia clone, and their histochemical, biochemical and functional characterizations were determined.

Results: Morphological changes into fibroblastoid stromal cells were observed in AML with t (11; 19) (p23; p13.1), M4E, Ph-positive biphenotype and CML (chronic and myeloid blast phase) cases but neither in ALL nor in CML-lymphoid blast cases. The generated fibroblastoid cells had enough functions equal to those of the normal bone marrow fibroblasts on their molecular expression (CD106, fibronectin), production of cytokines (VEGF, IL-7) and giving the activity of proliferation to normal hematopoietic cells. These cells maintained their characteristics observed in the original leukemia blasts (expression of CD13, CD33 and myeloperoxidase), which cells also expressed CD 34 and 133. Leukemia blasts proliferated extensively when cultured on the expanded fibroblastoid cells derived from leukemia blasts.

Discussion: These results indicate that leukemia blasts can create their own microenvironment for proliferation.
Bone Marrow Stroma-Produced TGF-beta1 Confers Chemoresistance of Leukemic Cells.
Authors: Tabe Yoko , Xu Yuanyuan , Jin Linhua , Andreeff Michael , Konopleva Marina
Published: April 2020

TGF-beta1 is secreted at high levels by bone marrow stromal cells and osteoblasts, however its role in leukemia/stroma interactions is unknown. We investigated the effects of TGF-beta1 on survival and proliferation of human monoblastic U937 cells. First, the production of TGF-beta1 by bone marrow-derived mesenchymal stem cells (MSCs) and by U937 cells was analyzed by ELISA. U937 cells secreted approximately 0.1 ng/ml of TGF-beta, while TGF-beta1 level was over 20-fold higher in MSC conditioned medium (2.1±1.7 ng/ml at 72 hours). When U937 cells were co-cultured with MSC, TGFbeta concentration was 2.2±0.2 ng/ml. Hence, we selected 2 ng/ml concentration of recombinant TGF-beta to examine the effects on survival and proliferation of U937 cells exposed to chemotherapy or serum starvation. TGF-beta1 significantly reduced Ara-C or serum withdrawal-induced apoptosis of U937 cells in particular when U937 cells were co-cultured with MSCs (AnnexinV(+)%;U937 alone, control 34.5±8.4, TGF-beta1 18.4±4.5, Ara-C 88.6±3.0, Ara-C/TGF-beta1 60.4±8.0, p=0.04; U937 co-cultured with MSC, control 19.4±2.8, TGF-beta1 3.5±1.0, Ara-C 69.0±3.6, Ara-C+TGF-beta1 24.9±3.3, p=0.01). Treatment with rhTGF-beta1 also enhanced percentage of cells in G0/1 cell cycle phase hence promoting quiescence (G0/G1 phase; control 50.1±6.7, TGF-beta1 63.8±5.5, p=0.04). Most importantly, inhibition of TGF-beta1 by neutralizing antibody (TGF-NA) significantly diminished the pro-survival effects of rhTGF-beta1 in serum-deprived U937 cells co-cultured with MSC (AnnexinV(+)%; U937 alone, control 27.9±9.0, TGF-beta1 20.1±3.3, TGF-NA 21.8±2.2, TGF-beta1+ TGF-NA 18.9±4.1; U937 co-cultured with MSC, control 22.2±1.9, TGF-beta1 10.9±2.5, TGF-NA 29.6±5.1, TGF-beta1+ TGF-NA 20.2±0.9, p<0.001). Dissection of molecular mechanisms demonstrated that TGF-beta1 induced anti-apoptotic Bcl-2 protein expression in U937 cells. Furthermore, concomitant exposure of U937 cells to rhTGF-beta1 in MSC co-cultures results in activation of AKT signaling and pro-survival phosphorylation of Bcl-2, implicating their role in anti-apoptotic effects of TGF-beta1. Taken together, these findings demonstrate that TGF-beta1 secreted by BM stroma cells promotes survival of U937 cells and confers chemoresistance of leukemic cells within the bone marrow microenvironment. Since small molecule inhibitors of TGF-beta signaling are undergoing final steps of pre-clinical development, this approach may represent a viable strategy to enhance efficacy of chemotherapy in AML.
Patulin: A Small Molecule Mycotoxin Possessing Potent Selective In Vitro Cytotoxicity Against Malignant T-Cells.
Authors: Rollins Margo R. , Hessel Alex , Rosenthal Hilary , Waller Ned
Published: April 2020

Background: Peripheral T-cell lymphomas have no established standard therapy, and clinical outcomes are inferior to those seen in patients with diffuse large B-ell lymphoma. Mycotoxins are structurally diverse secondary metabolites of fungi with immunosuppressive activity. Mycotoxins are believed to kill T-cells by covalently bonding to intermediates in the p38 map kinase pathway. We hypothesized that mycotoxins may have selective cytotoxicity against malignant T-cells.

Method: The cytotoxicity of Gliotoxin and Patulin were tested against normal peripheral blood mononuclear cells and malignant hematopoietic cell lines. Growth inhibition was assessed using an MTT assay and cytotoxicity was measured using flow cytometry following staining with 7AAD and annexin. Five leukemia/lymphoma cell lines: K562, KG-1, HL60, Jurkat, HL60, and MJ were grown in log phase culture. Malignant and normal mononuclear cells were incubated with serial dilutions of either Patulin or Gliotoxin for 24 hours. The concentration of drug producing half maximal cytotoxicity (ED50) was determined for each compound and each target cell population. Multiple replicates of this experiment were run 2 to 4 times and average values for experiments can found in the graphs below.

Results: Gliotoxin and Patulin were cytotoxic to normal and malignant hematopoietic cells at micromolar concentrations in the flow cytometry and MTT assays. Normal T-cells were more sensitive than tumor cells to the cytotoxic and growth inhibitory effects of Gliotoxin (Figure 1). In contrast, Patulin was selectively cytotoxic against malignant T-cell lines, with an ED50 of 0.5 μM for Jurkat and 5 μM for MJ compared to and ED50 of > 50 μM with normal T-cells. Low micromolar concentrations of Patulin produced more than 50% cell death in the Jurkat T-cell line with less than 20% killing of normal T-cells. CD11c+ type I dendritic cells were equally sensitive to both Patulin and Gliotoxin.

Conclusion: Gliotoxin and Patulin are cytotoxic against normal and malignant hematopoietic cells at micromolar concentrations. The selective cytotoxicity of Patulin on malignant T-cell lymphoma and leukemia cell lines suggests potential therapeutic activity in T-cell malignancies. The action of Patulin on intermediaries in the p38 map kinase pathway in normal and malignant T-cells will be discussed.

Glioitoxin Dose Response Curves Glioitoxin Dose Response Curves Patulin Dose Response Curves Patulin Dose Response Curves
The FRA12E Fragile Site Is Localized within the SMRT Gene at 12q24.
Authors: Coignet Lionel , Dolcce Jean , Ghoshal Pushpankur , Nganga Alain , Johnson Timothy , Foreman Heather , Moran-Guiati Joseph , Houde Christiane
Published: April 2020

The Silencing Mediator of retinoid and Thyroid hormone (SMRT) is a transcription co-repressor whose association with nuclear receptors both in solution and bound to DNA response elements is destabilized by ligand. SMRT and a related co-repressor N-CoR are recruiting a transcriptional repression complex, that contains sin3A/B protein and histone deacetylases (HDAC1/2) to nuclear receptors. It is now well established that SMRT can interact with non-receptor proteins such as BCL6, for its repression activities. The ability of the “silencing complex” to deacetylate histones results in a condensed chromatin state that is inhibitory to transcription. We have previously shown that SMRT was localized at chromosome 12q24. In addition, down-regulation of the SMRT protein due to 12q24 rearrangements was recurrently associated with NHL transformation, supporting a tumor suppressor function for SMRT (Cancer Res, 65(11):4554–4561, 2005). We further investigated the reasons why the 12q24 region and SMRT was targeted by chromosomal rearrangements. Genetic breakage is one mechanism by which functional loss of tumor suppressor gene activity may occur. Chromosomal locations in which genetic breakage may be induced are known as fragile sites. Fragile sites have been shown to be involved in some malignancies in which the fragile site lies within known genes, such as the FHIT gene (chromosome 3p) in lung cancer, and where small deletions are consistently observed on chromosome 3. Two fragile sites exist on the long arm of chromosome 12. FRA12B is located at 12q24.13 and FRA12E has been located at 12q24.2–3. Interestingly the FRA12E region corresponds to the site of SMRT (12q24.2). To assess whether the FRA12E fragile site is localized within the SMRT gene, we studied normal lymphocytes cultured in the presence of aphidicolin (an inducer of fragile sites) and metaphase chromosomes were subsequently prepared following classical cytogenetics protocols. These preparations were then subjected to fluorescence in situ hybridization (FISH) using a set of SMRT-specific RPCI BAC clones. Using this approach, we were able to demonstrate that the FRA12E fragile site is localized within the SMRT gene. We further characterize several lymphoblastoid cell lines that were either carrier or not of the FRA12E fragile site. This provides us with a mechanistic explanation for recurrent 12q24 rearrangements seen in transformed NHLs. The FRA12E-carrier lymphoblastoid cell lines will provide us the opportunity to characterize the structure of this fragile site.
Duplication and Amplification of AML1 in a Biphenotypic Acute Leukemia: Mechanism in the Formation of Double Minute Chromosome?.
Authors: Benattar Laurence K. , Bouffard Laurence , Coignet Lionel J.
Published: April 2020

Amplification of AML1 has been reported in pediatric acute lymphoblastic leukemia (ALL): it occurs mainly older in older children (5 to 14 years-old) as well as in CD10 positive B ALL and a few cases of acute myeloid leukemia. The presence of double minute chromosomes (DMs) may be one of the mechanisms for oncogene up-regulation. It is a rather rare event in hematological malignancies and is generally associated with a poor prognosis. Until now, DMs have been described mainly in AML and involve mainly amplification of c-MYC and MLL.

We report here the case of a 14-year-old boy with CD10 negative biphenotypic acute leukemia (B + M).

Bone marrow cytogenetic analysis revealed a (47, XY) clone with one normal chromosome 21 and two small markers consisting of chromosome 21 material as the sole abnormality. The two markers were consistent with different forms: small acrocentric, small metacentric and appear to be a mixture of those forms until resembling double minute.

Fluorescence in situ hybridization (FISH) was performed using the TEL-AML1 ES probe. Metaphase FISH showed one normal signal, one tandem signal and a third signal with an apparent variable structure. In interphase nuclei, the hybridization showed from 2 to >6 signals without neither tandem duplication nor clustered signals in an area within the interphase nuclei as previously reported in B-ALL.

In our case, we hypothesize that the different forms of the “derivative/deleted” chromosome 21 lead to form DMs containing the AML1 gene. The mechanism seems to be multistep chromosomal rearrangements with formation of an iso-chromosome 21 and deletion of part of chromosome 21.

To our knowledge, it is the first case of biphenotypic AL reported with AML1 amplification and a probable double minute mechanism.
Flow Cytometric Detection of Human Telomerase Reverse Transcriptase Expression in CD34 Positive Precursor Fraction in Myelodysplastic Syndrome Bone Marrow.
Authors: Handa Hiroshi , Matsushima Takafumi , Tsukamoto Norifumi , Karasawa Masamitsu , Irisawa Hiroyuki , Saito Takayuki , Yokohama Akihiko , Yamane Arito , Murakami Hirokazu
Published: April 2020

Telomerase activity has been found in most common cancers indicating that telomerase detection may be a useful marker in cancer diagnosis. For detection of telomerase activity and the expression of associated genes in cells, TRAP assay and RT-PCR are customarily used. Immunohistochemical detection of hTERT is useful to detect telomerase-positive cells in a background of non- cancerous cells. We developed a method for the detection of intra-nuclear hTERT protein, in a sub-population of hematopoietic cells, using concurrent staining cell surface antigen and multi color flow cytometry. Human leukemia and myeloma cell lines showed 100% positivity, whereas neutrophils of normal subjects showed 0% positivity, it is consistent with telomerase activity assessed by TRAP assay (r=0.71, p<0.0001) and previous observations. Then we applied this method to analyze hTERT expression in myelodysplastic syndrome (MDS). Forty MDS patients samples were obtained, 36 patients were diagnosed as low risk MDS (RA), 14 patients were diagnosed as high risk MDS (RAEB or RAEB-t) according to FAB classification. All samples were acquired after informed consent was obtained from the patients.

Expression of hTERT protein was higher in CD34-positive blast-gated cells than CD34-negative blast-gated cells. The percentage of the CD34+ cells expressing hTERT ranged from 9.66% to 90.91% in low risk MDS patients, whereas from 50.46% to 97.68% in high risk MDS. The expression level was higher in the high risk group compared to that in the low risk group in MDS (p=0.0054, p=0.0084). This observation implied that telomerase up-regulation and hTERT expression were important for disease progression and could be a marker of more advanced disease. In subsets of MDS and AML bone marrow specimens obtained from these patients, we examined the hTERT expression in CD34+/CD38 high cells and CD34+/CD38 low cells containing stem cell fraction. Of interest, some of the patients showed higher expression of hTERT in CD34+/CD38 low cells than in CD34+/CD38 high cells. This observation is inconsistent with previous reports describing normal bone marrow hematopoietic cell findings. We speculated that this phenomenon could be a marker of MDS abnormality and that telomerase up-regulation may be initiated in the more primitive precursor fraction containing hematopoietic stem cells during the disease progression. Telomerase studies may be useful for definition of the risks associated with disease severity. Multi-parameter nature of flow cytometry and its ability to identify cellular sub-populations will facilitate a fuller understanding of the mechanisms of activation of telomerase.
Changes in DNA Methylation of Repetitive Elements during the Progression of Chronic Myelogenous Leukemia.
Authors: Choi Si Ho , Byun Hyang-Min , Jiang Jia Yi , Garcia-Manero Guillermo , Yang Allen S.
Published: April 2020

Epigenetic changes are a common finding in cancer, and promoter hypermethylation of tumor suppressor genes leads to aberrant gene silencing. In contrast to gene promoter hypermethylation, global hypomethylation is also a well-described phenomenon in cancer. Measurement of total 5-methylcytosine in the DNA show decreases in cancer despite increases in gene specific methylation. This discrepancy can be accounted for by the decrease of DNA methylation found in DNA repetitive elements, which make up approximately over half of the human genome. Decreases in repetitive element DNA methylation are a sensitive surrogate marker of global methylation, and have been used to measure DNA methylation changes induced by chemotherapy, dietary folate and environmental exposure. Decreases in LINE1 methylation have been described in numerous human cancers. In CML decrease in LINE1 methylation has been associated with progression to blast crisis. We investigated the DNA methylation changes of DNA repetitive elements in CML during the progression of CML from chronic phase to accelerated phase and finally blast crisis. We used a previously described assay (Yang et al. 2004) employing Bisulfite-PCR Pyrosequencing to quantitatively study the DNA methylation changes associated with the LINE1, Alu, D4Z4, and NBL-2 repetitive elements (See table).

LINE1 and Alu hypomethylation was significantly associated with the progression of CML. Surprisingly, we found that NBL-2 and D4Z4 hypermethylation significantly increased during the progression of CML. Interestingly, these two repetitive elements were found to be specifically hypomethylated in immunodeficiency, centromeric instability and facial abnormalities (ICF) syndrome which is caused by a germline mutation of DNA methyltransferase 3b (DNMT3b) (Kondo T et al, 2000). This result suggests that DNMT3b may be involved in the progression of CML.

DNA methylation (Mean ± S.E.M) Repetitive Elements Normal Chronic Phase Accelerated Phase Blast Crisis LINE1 32.4 ± 1.3 29.3 ± 0.6 27.8 ± 0.8 28.3 ± 0.8 Alu 79.4 ± 1.1 77.9 ± 0.6 77.6 ± 0.9 77.5 ± 0.8 D4Z4 47.9 ± 2.9 49.0 ± 1.1 52.9 ± 1.4 57.5 ± 2.0 NBL-2 71.7 ± 2.2 71.7 ± 0.7 75.9 ± 1.3 77.2 ± 1.6
Hydroxyurea with Azacitidine or Decitabine in Combination Is Antagonistic on DNA Methylation Inhibition.
Authors: Choi Si Ho , Byun Hyang-Min , Kwan Jennifer , Yang Allen S.
Published: April 2020

5-azacytidine (azacitidine, Vidaza) and 5-aza-2′deoxycytidine (decitabine, Dacogen) are cytidine analogs currently used to treat myelodysplastic syndrome and as an investigational agent to treat acute myelogenous leukemia, sickle cell disease and other cancers. Decitabine is phosphorylated to a triphosphate form and incorporated into DNA where it can covalently trap DNA methyltransferase. Azacitidine is also phosphorylated to a triphosphate form, but requires reduction from a ribonucleotide to a deoxyribonucleotide by ribonucleotide reductase (RR) before incorporation into DNA. Hydroxyurea (Hydrea) is a drug routinely used to treat patients with leukemia and sickle cell disease. Hydroxyurea inhibits cell proliferation by inhibiting RR. Inhibition of RR leads to a depletion of nucleotide pools and cell cycle arrest in S-phase. Therefore, we hypothesized that the combination of hydroxyurea with azacitidine would be antagonistic to the ability of azacitidine to inhibit DNA methylation, and synergistic with decitabine in inhibiting DNA methylation. Hydroxyurea inhibits RR, which could block the conversion of azacitidine from a ribonucleotide to a deoxyribonucleotide, and therefore prevent its incorporation into DNA. In contrast, decitabine could increase its incorporation into DNA by taking advantage of a depletion of other deoxynucleotides induced RR inhibition by hydroxyurea. In order to test this hypothesis, we treated HL-60 (leukemia) and T24 (bladder cancer) cell lines with hydroxyurea in combination with either decitabine or azacitidine. DNA methylation was quantitated using Bisulfite-PCR Pyrosequencing of LINE-1 DNA repetitive elements, the MAGE-A1 gene and the p16 gene promoter region. We clearly showed that hydroxyurea at concentrations above 0.5 mM completely blocked the ability of azacitidine to inhibit DNA methylation (IC50=0.36 ± 0.04 mM). Surprisingly, we found that hydroxyurea at concentrations above 0.5mM also completely inhibited the effect of decitabine on DNA methylation (IC50=0.24 ± 0.06 mM). Further investigation showed that inhibition of the ability to block DNA methylation correlated with inhibition of the cell cycle. Using flow cytometry we clearly show that the antagonistic effect of hydroxyurea on DNA methylation inhibitors correlates with the arresting of cell division in S-phase. Aphidicolin, which inhibits DNA polymerase, also blocked the cell cycle and the ability of azacitidine and decitabine to inhibit DNA methylation. Thus, azacitidine and decitabine are S-phase specific drugs and are antagonized when used in combination with drugs that arrest the cell cycle. However, we also show that sequential treatment of hydroxyurea followed by azacitidine or decitabine could be used to avoid this antagonism. Therefore, due to the antagonistic effect of hydroxyurea with the mechanistic inhibitors of DNA methylation, azacitidine and decitabine, we recommend sequential combination treatment strategy.
Expression of LAT and PAG Adaptor Molecules in Blasts of Childhood Acute Lymphoblastic Leukaemias and in Normal B- and T-Precursors.
Authors: Burjanivova Tatiana , Vaskova Martina , Kalina Tomas , Stary Jan , Trka Jan , Zuna Jan
Published: April 2020

Despite the progress in leukaemia research, genetic background of some acute lymphoblastic leukaemias (ALL) is unknown. In the aetiopathogenesis of ALL the transmembrane adaptor proteins (including LAT and PAG) could play an important role as these proteins are key mediators of signalization cascades in lymphocytes.

We analysed expression of PAG and LAT at the mRNA level by real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and at the protein level by flow cytometry. In our study we assessed the expression levels in 10 patients with T-ALL and in 10 patients with B-cell precursor ALL (BCP-ALL). All analysed samples had a high percentage of malignant blasts (70–97%, median 91%). Sorted and unsorted bone marrow/peripheral blood samples of healthy volunteer donors were used as controls. To determine the PAG and LAT expression levels in normal immature thymocytes we analysed also sorted cells from the normal thymuses taken from children undergoing heart surgery. We used various differentiation markers of both B- (CD10, CD19, and CD20) and T-lineage (CD1a, CD3, CD4, CD7, CD8) for sorting to compare the leukaemic samples with the most adequate control in terms of maturation status of the cells. Informed consents were obtained from all volunteer donors or their guardians.

Our data show that the expression of PAG at both protein and mRNA level during the physiological B-cell maturation is decreasing. On the contrary, PAG expression during the T-cell development and also the levels of LAT during the B- and T-cell maturation are increasing, however, the levels of LAT mRNA in B-precursors are very weak.

In childhood BCP-ALL the expression of PAG on protein and mRNA level reflects the situation in corresponding non-malignant precursors with the exception of the TEL/AML1 positive leukaemias that show tendency to the overexpression of PAG. In the T lineage ALL we found a significantly lower expression of PAG and higher expression of LAT mRNA ((p<0.0001 and p=0.03, respectively, for mature T-ALL) compared to sorted thymocytes with corresponding immunophenotype. The LAT protein in BCP-ALL blasts is expressed very weakly or there is no expression at all. Also the levels of LAT mRNA are even lower than in the physiological BCP counterparts.

We present the first study assessing changes in the expression of two important adaptor molecules at both mRNA and protein levels during the physiological maturation of lymphocyte precursors. Moreover, we demonstrate changes in the pattern of expression in childhood ALL and suggest that the aberrant transmission or processing of signal through the immunoreceptors of lymphoid precursors might play an important role in the aetiopathogenesis of the disease.
Absence of JAK2 V617F Activating Mutations in Children with Acute Megakaryoblastic Leukemia with and without Down Syndrome.
Authors: Zwaan Christian M. , Broekhuis Mathilde J.C. , Langebrake Claudia , Goemans Bianca F. , Kaspers Gertjan J.L. , Pieters Rob , Reinhardt Dirk , den Boer Monique L.
Published: April 2020

Activating mutations at codon 617 of the Janus-2 tyrosine kinase (JAK2 V617F) have recently been described in hematological malignancies. In adult acute myeloid leukemia (AML), the reported frequencies vary, and JAK2 V617F mutations have mainly been detected in secondary AML following a myeloproliferative disorder. In adult de novo AML, the mutation was less frequent, and detected in 2/11 (18%) acute megakaryoblastic leukemia (FAB M7) samples (Jelinek et al., Blood 2005), and occasionally in other FAB-types.

This prompted us to analyze a cohort of pediatric AML FAB M7 samples for this particular mutation. In children, at least 3 different subsets of AML M7 can be identified: infants with AML M7 characterized by t(1;22)(p13;q13), older children with random cytogenetic aberrations, and myeloid leukemia of Down syndrome (DS ML). DS ML is often preceded by transient myeloproliferative disease (TMD), hence we also screened TMD samples to detect whether JAK2 V617F mutations would be involved in clonal evolution from TMD to DS ML. To exclude germ-line mutations in DS, we tested normal mononuclear bone marrow cells (NBMC) from children with DS. These NBMC were obtained from a sternal aspirate from children undergoing cardiac surgery, after informed consent was obtained. Genomic DNA was harvested from leukemic cells, and JAK2 exon 12, including the intron-flanking regions, was amplified and sequenced to screen for the JAK2 V617F mutation. As a positive control for the JAK2 V617F mutation, we used HEL 92.1.7 cells (an erythroleukemic cell line). In a dilution experiment we could still detect the mutation, using direct sequencing, if 10% HEL/JAK2 mutated cells were mixed with 90% wild-type control cells. We tested 49 samples, comprising of 9 NBMC, 11 TMD, 14 DS-ML M7, 11 non-DS AML M7 and 4 relapsed non-DS AML M7 samples (including 2 initial diagnosis-relapse pairs). The median age of the TMD cohort was 3 days, for DS-ML children 1.9 years (range 0.9–3.8 yrs), and for non-DS AML 1.5 years (range 1.2–13.7 yrs). The median white blood cell count for TMD was 25.8x109/l, for DS-ML 13.8x109/l, and for non-DS AML 12.4x109/l. Cytogenetic data were available in 5/11 non-DS AML cases only, which showed no cases with a t(1;22). No JAK2 V617F mutations were detected in any of the clinical samples. We conclude that the role of JAK2 V617F mutations in pediatric DS and non-DS acute megakaryoblastic leukemia is limited at best. However, we were not able to screen the subgroup of non-DS AML cases with t(1;22).