Human EVI9, a Homologue of the Mouse Myeloid Leukemia Gene, Is Expressed in the Hematopoietic Progenitors and Down-Regulated during Myeloid Differentiation of HL60 Cells

Evi9, a common site of retroviral integration in BXH2 murine myeloid leukemias, encodes a C₂H₂ zinc finger protein and is overexpressed in these leukemic cells. To investigate a possible role of EVI9 in the human hematopoietic system, we isolated the cDNA clone of the human homologue. Human EVI9, located on the chromosome 2p13 region, contains an open reading frame of 797 amino acids that is 98.7% identical to the mouse protein. RT-PCR analysis of purified human hematopoietic cells showed that EVI9 is expressed in CD34-positive myeloid precursors, B cells, monocytes, and megakaryocytes, but only weakly in T lymphocytes, suggesting that EVI9 may play an important role in hematopoiesis. Furthermore, EVI9 was down-regulated during myeloid differentiation of HL60 cells induced by all-trans-retinoic acid, whereas the expression remained during monocytic differentiation induced by phorbol 12-myristate 13-acetate. These results indicate a distinct role for EVI9 in human hematopoietic cells and suggest that EVI9 may cause leukemia through inhibition of myeloid differentiation.     

Fluctuation of ROS regulates proliferation and mediates inhibition of migration by reducing the interaction between DLC1 and CAV-1 in breast cancer cells

The aim of our present study was to elucidate the effects of up-regulation and down-regulation of intracellular reactive oxygen species (ROS) level on proliferation, migration, and related molecular mechanism. Breast cancer cells were treated by catalase or H₂O₂. MTT, colony formation assay, and Hoechst/PI staining were used to evaluate proliferation and apoptosis. The level of intracellular ROS was measured by dichlorodihydrofluorescein diacetate probes. The ability of migration was detected by wound healing. Western blotting and coimmunoprecipitation (co-IP) were used to determine the expression of DLC1 and CAV-1 and their interaction. Our data indicated that up-regulation of intracellular ROS induced by H₂O₂ significantly inhibited proliferation and induced apoptosis accompanying G1 cell cycle arrest and elevated expression of p53. For cell migration, either up-regulation or down-regulation of ROS induced migration inhibition with reduction of interaction between DLC1 and CAV-1. Our results suggested that up-regulation of intracellular ROS inhibited proliferation by promoting expression of p53 and induced G1 cycle arrest and apoptosis. Fluctuation of ROS inhibited migration through reducing the interaction between DLC1 and CAV-1.     

Alcohol impairs the myeloid proliferative response to bacteremia in mice by inhibiting the stem cell antigen-1/ERK pathway

Enhancement of stem cell Ag-1 (Sca-1) expression by myeloid precursors promotes the granulopoietic response to bacterial infection. However, the underlying mechanisms remain unclear. ERK pathway activation strongly enhances proliferation of hematopoietic progenitor cells. In this study, we investigated the role of Sca-1 in promoting ERK-dependent myeloid lineage proliferation and the effects of alcohol on this process. Thirty minutes after i.p. injection of alcohol, mice received i.v. challenge with 5 × 10(7) Escherichia coli for 8 or 24 h. A subset of mice received i.v. BrdU injection 20 h after challenge. Bacteremia increased Sca-1 expression, ERK activation, and proliferation of myeloid and granulopoietic precursors. Alcohol administration suppressed this response and impaired granulocyte production. Sca-1 expression positively correlated with ERK activation and cell cycling, but negatively correlated with myeloperoxidase content in granulopoietic precursors. Alcohol intoxication suppressed ERK activation in granulopoietic precursors and proliferation of these cells during bacteremia. Granulopoietic precursors in Sca-1(-/-) mice failed to activate ERK signaling and could not increase granulomacrophagic CFU activity following bacteremia. These data indicate that Sca-1 expression promotes ERK-dependent myeloid cell proliferation during bacteremia. Suppression of this response could represent an underlying mechanism for developing myelosuppression in alcohol-abusing hosts with severe bacterial infection.     

Redox regulation of stem/progenitor cells and bone marrow niche

Bone marrow (BM)-derived stem and progenitor cell functions including self-renewal, differentiation, survival, migration, proliferation, and mobilization are regulated by unique cell-intrinsic and -extrinsic signals provided by their microenvironment, also termed the “niche.” Reactive oxygen species (ROS), especially hydrogen peroxide (H₂O₂), play important roles in regulating stem and progenitor cell functions in various physiologic and pathologic responses. The low level of H₂O₂ in quiescent hematopoietic stem cells (HSCs) contributes to maintaining their “stemness,” whereas a higher level of H₂O₂ within HSCs or their niche promotes differentiation, proliferation, migration, and survival of HSCs or stem/progenitor cells. Major sources of ROS are NADPH oxidase and mitochondria. In response to ischemic injury, ROS derived from NADPH oxidase are increased in the BM microenvironment, which is required for hypoxia and hypoxia-inducible factor-1α expression and expansion throughout the BM. This, in turn, promotes progenitor cell expansion and mobilization from BM, leading to reparative neovascularization and tissue repair. In pathophysiological states such as aging, atherosclerosis, heart failure, hypertension, and diabetes, excess amounts of ROS create an inflammatory and oxidative microenvironment, which induces cell damage and apoptosis of stem and progenitor cells. Understanding the molecular mechanisms of how ROS regulate the functions of stem and progenitor cells and their niche in physiological and pathological conditions will lead to the development of novel therapeutic strategies.     

Induction of apoptosis in a human leukemic cell line via reactive oxygen species modulation by antioxidants

In the human acute myeloid leukemia cell line M07e, the growth factor interleukin-3 (IL-3) induces ROS formation, positively affecting Glut1-mediated glucose uptake and cell survival. The effect of IL-3 and exogenous hydrogen peroxide on cell viability seems to be mediated through inhibition of the cell death commitment, as shown by apoptotic markers such as caspase activities, apoptotic nuclei, and changes in the amount of proteins belonging to the Bcl-2 family. The pivotal role of ROS is confirmed using various antioxidants, such as EUK-134, ebselen, TEMPO, and hydroxylamine probe. In fact, these antioxidants, acting through different mechanisms, decrease glucose transport activity and cell proliferation activated by IL-3 or by low concentrations of hydrogen peroxide. Moreover, antioxidants foster programmed cell death commitment, as shown by the cited apoptotic parameters. EUK-134, a combined superoxide dismutase/catalase mimetic, opposes the effects of IL-3 and H₂O₂, decreasing phosphorylation levels of signaling enzymes such as Akt, Src tyrosine kinase, and ERK. Results show that ROS production induced by IL-3 can protect leukemic cells from apoptosis, the effect being counteracted by antioxidants. This mechanism may play an important role in supporting acute myeloid leukemia treatment, thus representing a novel therapeutic strategy.     

Gene Expression Profiling Identifies HOXB4 as a Direct Downstream Target of GATA-2 in Human CD34+ Hematopoietic Cells

Aplastic anemia is characterized by a reduced hematopoietic stem cell number. Although GATA-2 expression was reported to be decreased in CD34-positive cells in aplastic anemia, many questions remain regarding the intrinsic characteristics of hematopoietic stem cells in this disease. In this study, we identified HOXB4 as a downstream target of GATA-2 based on expression profiling with human cord blood-derived CD34-positive cells infected with control or GATA-2 lentiviral shRNA. To confirm the functional link between GATA-2 and HOXB4, we conducted GATA-2 gain-of-function and loss-of-function experiments, and HOXB4 promoter analysis, including luciferase assay, in vitro DNA binding analysis and quantitative ChIP analysis, using K562 and CD34-positive cells. The analyses suggested that GATA-2 directly regulates HOXB4 expression through the GATA sequence in the promoter region. Furthermore, we assessed GATA-2 and HOXB4 expression in CD34-positive cells from patients with aplastic anemia (n = 10) and idiopathic thrombocytopenic purpura (n = 13), and demonstrated that the expression levels of HOXB4 and GATA-2 were correlated in these populations (r = 0.6573, p<0.01). Our results suggested that GATA-2 directly regulates HOXB4 expression in hematopoietic stem cells, which may play an important role in the development and/or progression of aplastic anemia.     

HERG1 Currents in Native K562 Leukemic Cells

The human ether-a-go-go related gene (HERG1) K⁺ channel is expressed in neoplastic cells, in which it was proposed to play a role in proliferation, differentiation and/or apoptosis. K562 cells (a chronic myeloid leukemic human cell line) express both the full-length (herg1a) and the N-terminally truncated (herg1b) isoforms of the gene, and this was confirmed with Western blots and coimmunoprecipitation experiments. Whole-cell currents were studied with a tail protocol. Seventy-eight percent of cells showed a HERG1-like current: repolarization to voltages negative to −40 mV produced a transient peak inward tail current, characteristic of HERG1 channels. Cells were exposed to a HERG-specific channel blocker, E4031. Half-maximal inhibitory concentration (IC₅₀) of the blocker was 4.69 nm. The kinetics of the HERG1 current in K562 cells resembled the rapid component of the native cardiac delayed rectifier current, known to be conducted by heterotetrameric HERG1 channels. Fast and slow deactivation time constants at −120 mV were 27.5 and 239.5 ms, respectively. Our results in K562 cells suggest the assembling of heterotetrameric channels, with some parameters being dominated by one of the isoforms and other parameters being intermediate. Hydrogen peroxide was shown to increase HERG1a K⁺ current in heterologous expression systems, which constitutes an apoptotic signal. However, we found that K562 HERG1 whole-cell currents were not activated by H₂O₂.     

The proliferation index of specific bone marrow cell compartments from myelodysplastic syndromes is associated with the diagnostic and patient outcome

Myelodysplastic syndromes (MDS) are clonal stem cell disorders which frequently show a hypercellular dysplastic bone marrow (BM) associated with inefficient hematopoiesis and peripheral cytopenias due to increased apoptosis and maturation blockades. Currently, little is known about the role of cell proliferation in compensating for the BM failure syndrome and in determining patient outcome. Here, we analyzed the proliferation index (PI) of different compartments of BM hematopoietic cells in 106 MDS patients compared to both normal/reactive BM (n = 94) and acute myeloid leukemia (AML; n = 30 cases) using multiparameter flow cytometry. Our results show abnormally increased overall BM proliferation profiles in MDS which significantly differ between early/low-risk and advanced/high-risk cases. Early/low-risk patients showed increased proliferation of non-lymphoid CD34⁺ precursors, maturing neutrophils and nucleated red blood cells (NRBC), while the PI of these compartments of BM precursors progressively fell below normal values towards AML levels in advanced/high-risk MDS. Decreased proliferation of non-lymphoid CD34⁺ and NRBC precursors was significantly associated with adverse disease features, shorter overall survival (OS) and transformation to AML, both in the whole series and when low- and high-risk MDS patients were separately considered, the PI of NRBC emerging as the most powerful independent predictor for OS and progression to AML. In conclusion, assessment of the PI of NRBC, and potentially also of other compartments of BM precursors (e.g.: myeloid CD34⁺ HPC), could significantly contribute to a better management of MDS.