Experimental Hematology

Editorial Board

2010-08-01

Both Nramp1 and DMT1 are necessary for efficient macrophage iron recycling

2010-04-14

Objective: Divalent metal transporter 1 (DMT1) and natural resistance-associated macrophage protein 1 (Nramp1) are iron transporters that localize, respectively, to the early and late endosomal compartments. DMT1 is ubiquitously expressed, while Nramp1 is found only within macrophages and neutrophils. Our previous studies have identified a role for Nramp1 during macrophage erythrophagocytosis; however, little is known about the function of DMT1 during this process.Materials and Methods: Wild-type RAW264.7 macrophages (RAW), and those stably transfected with Nramp1 (RAW/Nramp1) were treated with either DMT1−small interfering RNA, or with ebselen, a selective inhibitor of DMT1.Results: Although macrophages lacking either functional DMT1 or Nramp1 experienced a moderate reduction in iron recycling efficiency, the ability of macrophages lacking both functional DMT1 and Nramp1 to recycle hemoglobin-derived iron was severely compromised. Compared to macrophages singly deficient in either DMT1 or Nramp1 transport ability, macrophages where DMT1 and Nramp1 were both compromised exhibited an abrogated increase in labile iron pool content, released less iron, and experienced diminished upregulation of ferroportin and heme-oxygenase 1 levels following erythrophagocytosis.Conclusions: These results suggest that although the loss of either Nramp1 or DMT1 transport ability results in minor impairment after erythrophagocytosis, the simultaneous loss of both Nramp1 and DMT1 iron transport activity is detrimental to the iron recycling capacity of the macrophage.

Distribution of dystrophin- and utrophin-associated protein complexes during activation of human neutrophils

Doris Cerecedo, Bulmaro Cisneros, Pablo Gómez, Iván J. Galván — 2010-04-30

Objective: Dystrophins, utrophins, and their associated proteins are involved in structural and signaling roles in nonmuscle tissues; however, description of these proteins in neutrophils remained unexplored. Therefore we characterize the pattern expression, and the cellular distribution of dystrophin and utrophin gene products and dystrophin-associated proteins (i.e., β-dystroglycan, α-syntrophin, and α-dystrobrevins) in relation to actin filaments in resting and activated with formyl-methionyl-leucyl-phenylalanine human neutrophils.Materials and Methods: Resting and fMLP-activated human neutrophils were analyzed by immunoblot and by confocal microscopy analysis. Immunoprecipitation assays were performed to corroborate the presence of protein complexes.Results: Immunoprecipitation assays and confocal analysis demonstrated the presence of two dystrophin-associated protein complexes in resting and activated neutrophils: the former formed by Dp71d/Dp71Δ110m and dystrophin-associated proteins (β-dystroglycan, α-syntrophin, α-dystrobrevin-1, and -2), while the latter contains Up400, instead of Dp71d/Dp71Δ110m, as a central component of the dystrophin-associated protein complexes (DAPC). Confocal analysis also showed the subcellular redistribution of Dp71d/Dp71Δ110m∼DAPC and Up400∼DAPC in F-actin−based structures displayed during activation process with fMLP.Conclusions: Our study showed the existence of two protein complexes formed by Dp71d/Dp71Δ110m or Up400 associated with DAPs in resting and fMLP-treated human polymorphonuclears. The interaction of these complexes with the actin cytoskeleton is indicative of their dynamic participation in the chemotaxis process.

MIR-23A microRNA cluster inhibits B-cell development

2010-04-16

Objective: The transcription factor PU.1 (encoded by Sfpi1) promotes myeloid differentiation, but it is unclear what downstream genes are involved. Micro RNAs (miRNAs) are a class of small RNAs that regulate many cellular pathways, including proliferation, survival, and differentiation. The objective of this study was to identify miRNAs downstream of PU.1 that regulate hematopoietic development.Materials and Methods: miRNAs that change expression in a PU.1-inducible cell line were identified with microarrays. The promoter for an miRNA cluster upregulated by PU.1 induction was analyzed for PU.1 binding by electrophoretic mobility shift and chromatin immunoprecipitation assays. Retroviral transduction of hematopoietic progenitors was performed to evaluate the effect of miRNA expression on hematopoietic development in vitro and in vivo.Results: We identified an miRNA cluster whose pri-transcript is regulated by PU.1. The pri-miRNA encodes three mature miRNAs: miR-23a, miR-27a, and miR-24-2. Each miRNA is more abundant in myeloid cells compared to lymphoid cells. When hematopoietic progenitors expressing the 23a cluster miRNAs were cultured in B-cell−promoting conditions, we observed a dramatic decrease in B lymphopoiesis and an increase in myelopoiesis compared to control cultures. In vivo, hematopoietic progenitors expressing the miR-23a cluster generate reduced numbers of B cells compared to control cells.Conclusions: The miR-23a cluster is a downstream target of PU.1 involved in antagonizing lymphoid cell fate acquisition. Although miRNAs have been identified downstream of PU.1 in mediating development of monocytes and granulocytes, the 23a cluster is the first downstream miRNA target implicated in regulating development of myeloid vs lymphoid cells.

AEE788 is a vascular endothelial growth factor receptor tyrosine kinase inhibitor with antiproliferative and proapoptotic effects in acute myeloid leukemia

2010-04-08

Objective: Aberrant activation of tyrosine kinase receptors is frequently observed in acute myelogenous leukemia (AML). Moreover, activating mutations of the fms-like tyrosine kinase 3 (FLT3) receptor can be found in approximately 30% of patients, thereby representing one of the most frequent single genetic alterations in AML. AEE788, a novel dual receptor tyrosine kinase inhibitor of endothelial growth factor and vascular endothelial growth factor (VEGF), is being studied in several solid tumors with remarkable success. It is not known, however, about the efficacy of this inhibitor in the treatment of AML. Therefore, we investigated the effect of AEE788 in the treatment of three human AML cell lines and seven AML patient samples.Materials and Methods: Cell survival in THP-1, MOLM-13, and MV4-11 cell lines (the two last harboring the FLT3/internal tandem duplication mutation) and AML blasts incubated with 0.5 to 15 μM AEE788 were quantified. We also studied the activation of VEGF/VEGF receptors loop, FLT3, and their downstream effectors (Akt, extracellular signal-regulated kinase, signal transducers and activators of transcription 5, and nuclear factor−κB).Results: Our data showed that AEE788 was a tyrosine kinase inhibitor of FLT3 activity and had antiproliferative and proapoptotic activity in AML-derived cell lines and AML blasts that presented phosphorylation of the FLT3 receptor. Consistently, in these cells AEE788 abrogated VEGF/VEGF receptors activation and the survival signaling pathways studied.Conclusion: Taken together, the activity of AEE788 might represent a promising new option of targeting FLT3 for the treatment of AML.

Aberrantly expressed CEACAM6 is involved in the signaling leading to apoptosis of acute lymphoblastic leukemia cells

Veronika Kanderová, Ondřej Hrušák, Tomáš Kalina — 2010-04-08

Objective: The aberrant expression of myeloid antigens on acute lymphoblastic leukemia (ALL) cells is a well-documented phenomenon. So far, there have been no reports of a functional consequence of this aberrant expression. The granulocytic marker carcinoembryonic antigen−related cell adhesion molecule 6 (CEACAM6, CD66c) is a GPI-anchored molecule that is reported to be the most frequently aberrantly expressed myeloid marker in ALL with a strong correlation with genotype.Materials and Methods: We mimicked CEACAM6 signaling in ALL cells by cross-linking with anti-CEACAM6 antibody. Next, we measured a response to CEACAM6 signaling by integrin subunits expression, integrin ligand binding, phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2), Akt, and p38 mitogen-activated protein kinase (MAPK) and apoptosis by flow cytometry.Results: Following CEACAM6 cross-linking in ALL cells, we detected Erk1/2, Akt, and p38 MAPK phosphorylation and integrin upregulation, as well as enhanced binding of integrin ligands (vascular cell adhesion molecule-1 [VCAM-1] and intercellular cell adhesion molecule-1 [ICAM-1]). However, CEACAM6 signaling resulted in an increase in apoptosis, unlike other GPI-anchored molecules, such as CD24.Conclusion: The present study is the first to demonstrate the functional consequences of CEACAM6 cross-linking in B-cell precursor ALL cells.

Detection of differential mitotic cell age in bone marrow CD34+ cells from patients with myelodysplastic syndrome and acute leukemia by analysis of an epigenetic molecular clock DNA signature

2010-04-16

Objective: Recently, the “epigenetic molecular clock hypothesis” linked increasing DNA methylation in a distinct CpG island in the cardiac-specific homeobox gene (CSX) gene to relative mitotic cell age. To determine mitotic cell age in hematopoietic cells of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients, we assessed differential CSX methylation patterns in these diseases vs age-adjusted healthy controls.Materials and Methods: We performed bisulfite pyrosequencing to analyze CSX methylation in CD34+ and bone marrow (BM) cells from 53 MDS, 62 AML, 77 ALL patients, and 37 controls.Results: Analysis of MDS CD34+ and BM cells revealed significantly increasing methylation of CSX in controls < MDS low-risk < MDS high-risk < AML. Furthermore, increased differences of CSX methylation between the CD34+ vs the unselected BM compartment were detected in matched MDS low-risk but not high-risk and AML samples. ALL samples displayed highly elevated CSX methylation levels as compared to controls.Conclusions: Assessment of mitotic cell age by CSX methylation analysis could reveal novel insights into the distinct progression of hematologic diseases.

Inhibition of mTORC1 by RAD001 (everolimus) potentiates the effects of 1,25-dihydroxyvitamin D3 to induce growth arrest and differentiation of AML cells in vitro and in vivo

2010-04-12

Objective: Differentiation-inducing therapy by agents such as 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) represents a useful approach for the treatment of acute myelogenous leukemia (AML). We previously showed that Gemini-23-yne-26,27-hexafluoro-D3 inhibited the proliferation of MCF-7 breast cancer cells in association with inhibition of the mammalian target of rapamycin (mTOR) signaling. This study explored the drug interaction of 1,25(OH)2D3 and rapamycin analog RAD001 (everolimus) in AML cells.Materials and Methods: Effects of RAD001 and 1,25-(OH)2D3 on the proliferation and differentiation of U937 cells were assessed by colony-forming assay and quantification of CD11b cell surface antigens and their endocytic capability, respectively. Effects of RAD001 and 1,25-(OH)2D3 on Akt/mTOR complex-1 (mTORC1) signaling and cell-cycle−related molecules were explored by Western blot analysis. The reporter gene and chromatin immunoprecipitation assays were employed to examine the effects of RAD001 and 1,25-(OH)2D3 on the promoter of the p21waf1 gene. U937 murine xenograft model was utilized to explore the effects of RAD001 and 1,25-(OH)2D3 in vivo.Results: RAD001 potentiated the ability of 1,25(OH)2D3 to induce growth arrest and differentiation of AML cells in parallel with downregulation of the levels of p-S6K and p-4E-BP1, substrates of mTORC1. In addition, RAD001 significantly enhanced 1,25(OH)2D3-mediated transcriptional activity of p21waf1 in association with increased levels of the acetylated forms of histone H3 and vitamin D receptor bound to the p21waf1 promoter in U937 cells. Moreover, RAD001 (3 mg/kg, every another day) significantly enhanced 1,25(OH)2D3-induced growth inhibition of U937 tumor xenografts in nude mice without adverse effects.Conclusions: Concomitant administration of 1,25(OH)2D3 and the mTORC1 inhibitor may be a promising treatment strategy for individuals with AML.

In vitro deprivation of CD8+CD57+T cells promotes the malignant growth of bone marrow colony cells in patients with lower-risk myelodysplastic syndrome

Zhang Zheng, Zhang Qianqiao, He Qi, Xu Feng, Chang Chunkang, Li Xiao — 2010-04-14

Objective: CD8+T lymphocytes have inhibitory effects on the proliferation of malignant clones in myelodysplastic syndrome (MDS). The exact CD8+T subset involved in the regulation of MDS and the target clones of CD8+T lymphocytes has not been studied. We investigated the effect of activated CD8+T (CD8+CD57+) lymphocytes on colony formation (in particular, malignant colony formation) during MDS in vitro.Materials and Methods: Bone marrow mononuclear cells (BMNCs) from a total of 59 MDS patients were subjected to magnetic-activated cell sorting to separate CD8+CD57+T lymphocytes. BMNCs were cultured without CD8+CD57+T cells or cocultured with a 1:4 ratio of CD8+CD57+T cells to study the association between stem/progenitor cell colony formation and the existence of activated CD8+ T cells, as well as the polarization of T cells towards Tc1. In addition, the fluorescence in situ hybridization method was used to detect bone marrow cells carrying abnormal karyotypes, and the proportion of abnormal cells among BMNCs was calculated before and after T-cell deprivation culture in vitro. Crossing cultures between MDS patients and normal volunteer was performed. The impact of effector CD8+T cells on the malignant growth of BMNCs was closely examined.Results: After deprivation of CD8+CD57+T cells, BMNCs from 33 MDS patients formed colonies in the culture media. The average number cells in the granulocyte and monocyte colony-forming units (CFU-GM) was 40.3/4 × 105, and the average number of cells in the erythroid colony-forming unit (CFU) was 10.4/4 × 105. These totals were significantly lower than those in the normal control group after deprivation of CD8+CD57+T cells (CFU-GM 83.4/4 × 105 cells, erythroid CFU 32.8/4 × 105 cells; p < 0.001). After add-back of CD8+CD57+ T cells (four times), none of the BMNCs cultures from any of the 59 MDS patients formed colonies in vitro. Additionally, in 33 MDS patients whose BMNCs formed colonies after T-cell deprivation, the bone marrow Tc1/Tc2 ratio was positively correlated with CFU-GM count (r = 0.443, p < 0.05). Crossing cultures indicated that CD8+CD57+ T cells from MDS patients cocultured with BMNC from normal donor did not show inhibition to colony-forming. In 15 MDS patients with abnormal karyotypes, deprivation of CD8+CD57+T cells significantly increased the proportion of abnormal cells from 43.8% to 56.3% in BMNC culture (p < 0.001).Conclusion: Effector CD8+T lymphocytes inhibit bone marrow hematopoiesis in MDS patients; target cells were primarily cells with abnormal karyotypes.

A potential activity of valproic acid in the stimulation of interleukin-3−mediated megakaryopoiesis and erythropoiesis

2010-04-09

Objective: Although the anticancer activities of histone deacetylase (HDAC) inhibitors have been studied, a role for HDAC in normal hematopoiesis has not been clearly defined. Previous studies have shown that the potent HDAC inhibitor FK228 stimulates interleukin (IL)-3−mediated erythropoiesis. Here, we examined whether the widely used valproic acid (VPA) affects megakaryopoiesis as well as erythropoiesis.Materials and Methods: CD34+ cells were incubated in serum-free or serum-containing cultures with cytokines, with or without VPA.Results: In the serum-free cultures containing IL-3+stem cell factor (SCF), VPA significantly increased generation of CD61+GPA− megakaryocytic and a CD61+GPA+ mixture of megakaryocytic and erythroid precursors from CD34+ hematopoietic precursors at a pharmacological concentration (100 μg/mL). The increase in generation of megakaryocytic and erythroid precursors by VPA was confirmed by replating cultured cells with thrombopoietin+SCF and erythropoietin+SCF, respectively. VPA was as potent as FK228. In cultures with granulocyte-macrophage colony-stimulating factor+SCF, where CD61−GPA+ erythroid precursors were mostly developed, VPA mainly enhanced the generation of CD61−GPA+ erythroid precursors. In serum-containing cultures, only low numbers of CD61+ or GPA+ cells were developed with IL-3+SCF. Nevertheless, a substantial number of these cells were generated with VPA. Furthermore, these stimulating effects of VPA were observed by incubating CD34+ cells from patients with myelodysplastic syndrome. Quantitative reverse transcription polymerase chain reaction showed that VPA enhanced GATA-2, but not GATA-1, messenger RNA expression with IL-3+SCF.Conclusions: These results indicate a novel role for VPA in enhancing the potential of IL-3 to stimulate megakaryopoiesis as well as erythropoiesis and suggest a new therapeutic approach of epigenetic therapy for hematological disease.

FET family proto-oncogene Fus contributes to self-renewal of hematopoietic stem cells

2010-04-21

Objective: Fus is the gene for a member of the FET family of RNA-binding proteins often involved in chromosomal translocations to generate oncogenic fusion genes in human cancers. Fus participates in multiple cellular functions, including RNA processing and transport, transcriptional regulation, and genome integrity. However, its role in hematopoiesis remains obscure. In this study, we examined its role in the self-renewal of hematopoietic stem cells (HSCs).Materials and Methods: HSCs in Fus−/− fetal livers were analyzed for proliferative capacity in vitro and long-term repopulating capacity in recipient mice. Radiation sensitivity of Fus−/− HSCs was evaluated in recipient mice repopulated by Fus−/− fetal liver cells.Results: Fus−/− fetal livers developed normally, except for a mild reduction in numbers of hematopoietic stem and progenitor cells compared to wild-type. The proliferation and differentiation of Fus−/− hematopoietic progenitors were normal in vitro. However, the number of colony-forming cells present in long-term cocultures of Fus−/− hematopoietic progenitors and stromal cells was significantly reduced. Fus−/− HSCs had an impaired long-term repopulating capacity and failed to repopulate in tertiary recipient mice. Fus−/− HSCs were highly susceptible to radiation both in vitro and in vivo and showed retardation of radiation-induced DNA damage repair.Conclusion: Our findings define Fus as a novel regulator of self-renewal and radioprotection of HSCs and also implicate it in stress-resistance and maintenance of the genomic integrity of HSCs.