Growth Of Hematopoietic Progenitor Cells Research Articles

The role of the GPR91 ligand succinate in hematopoiesis

Regulation of cellular metabolism by the citric acid cycle occurs in the mitochondria. However, the citric acid cycle intermediate succinate was shown recently to be a ligand for the G-protein-coupled receptor GPR91. Here, we describe a role for succinate and its receptor in the stimulation of hematopoietic progenitor cell (HPC) growth. GPR91 mRNA and protein expression were detected in human bone marrow CD34+ progenitor cells, as well as in erythroid and megakaryocyte cultures and the erythroleukemic cell line TF-1. Treatment of these cell cultures with succinate resulted in increased proliferation rates. The proliferation response of TF-1 cells was pertussis toxin (PTX)-sensitive, suggesting a role for Gi signaling. Proliferation was also blocked when TF-1 cells were transfected with small interfering RNA specific for GPR91. Succinate stimulated activation of the Erk MAPK pathway and inositol phosphate accumulation in a PTX-sensitive manner. Pretreatment of TF-1 cells with the Erk1/2 kinase (MEK) inhibitor PD98059 blocked the proliferation response. Succinate treatment additionally protected TF-1 cells from cell death induced by serum deprivation. Finally, in vivo administration of succinate was found to elevate the levels of hemoglobin, platelets, and neutrophils in a mouse model of chemotherapy-induced myelosuppression. These results suggest that succinate-GPR91 signaling is capable of promoting HPC development.

14-3-3γ Is Stimulated by IL-3 and Promotes Cell Proliferation

IL-3 plays important roles in the growth and survival of hematopoietic progenitor cells, processes modeled in studies of the IL-3-dependent cell line Ba/F3. To gain insights into molecular mechanisms governing cell fate, we examined the patterns of proteins up-regulated following stimulation of Ba/F3 cells with IL-3. Through two-dimensional electrophoresis and proteomics-based approaches, we identified 11 proteins. Of these, expression of 14-3-3gamma was significantly increased by IL-3 stimulation at both the transcriptional and translational levels. 14-3-3gamma overexpression in Ba/F3 cells abrogated dependence on IL-3 and was associated with activation of PI3K and MAPK signaling cascades, suggesting that the functions of 14-3-3gamma in normal hematopoietic progenitors are to promote survival and growth through the activation of distinct signaling pathways. Additionally, the up-regulation of Bax and Bad was seen with the ablation of 14-3-3gamma, resulting in cell death. These results indicate that deregulated expression of 14-3-3gamma may contribute to malignant transformation, possibly providing a new target for therapeutic intervention in hematopoietic neoplasms.

HIV-1 Infection of Bone Marrow Hematopoietic Progenitor Cells and Their Role in Trafficking and Viral Dissemination

Patients with HIV-1 often present with a wide range of hematopoietic abnormalities, some of which may be due to the presence of opportunistic infections and to therapeutic drug treatments. However, many of these abnormalities are directly related to HIV-1 replication in the bone marrow (BM). Although the most primitive hematopoietic progenitor cells (HPCs) are resistant to HIV-1 infection, once these cells begin to differentiate and become committed HPCs they become increasingly susceptible to HIV-1 infection and permissive to viral gene expression and infectious virus production. Trafficking of BM-derived HIV-1-infected monocytes has been shown to be involved in the dissemination of HIV-1 into the central nervous system (CNS), and it is possible that HIV-1 replication in the BM and infection of BM HPCs may be involved in the early steps leading to the development of HIV-1-associated dementia (HAD) as an end result of this cellular trafficking process. In addition, the growth and development of HPCs in the BM of patients with HIV-1 has also been shown to be impaired due to the presence of HIV-1 proteins and changes in the cytokine milieu, potentially leading to an altered maturation process and to increased cell death within one or more BM cell lineages. Changes in the growth and differentiation process of HPCs may be involved in the generation of monocyte populations that are more susceptible and/or permissive to HIV-1, and have potentially altered trafficking profiles to several organs, including the CNS. A monocyte subpopulation with these features has been shown to expand during the course of HIV-1 disease, particularly in HAD patients, and is characterized by low CD14 expression and the presence of cell surface CD16.

Nocturnal Hemodialysis Improves Erythropoietin Responsiveness and Growth of Hematopoietic Stem Cells

Nocturnal home hemodialysis (NHD) is associated with an increase in hemoglobin level. We hypothesized that NHD enhances the removal of toxins of hematopoietic progenitor cells (HPCs), thereby improving HPC growth and function. Among 16 patients with ESRD, 2 mo of NHD nearly doubled Kt/V per session and significantly lowered both parathyroid hormone levels and serum phosphate concentration. In addition, treatment with NHD improved hemoglobin levels from 113 +/- 3 to 125 +/- 4 g/L (P = 0.03) without altering erythropoietin requirements or iron status. To assess whether NHD may enhance removal of HPC toxins, we collected paired plasma samples from the same patient during treatment with conventional HD and NHD. In vitro, growth of erythroid (BFU-E) and granulocytic (CFU-GM) colonies was superior when cultured with NHD plasma compared with conventional HD plasma. Differential gene expression profiles obtained from peripheral blood and HPC colonies revealed similar upregulation of genes responsible for HPC mobilization and growth and production of red blood cells. In conclusion, the enhanced clearance by NHD is associated with an improvement in HPC growth and a coordinated increase in expression of genes relevant to production of red blood cells.

ChIP-Chip in Primary AML Blasts Identifies Peroxiredoxin-II as An Epigenetically Silenced Tumor Suppressor.

Epigenetic silencing of tumor suppressors is a frequent event in leukemogenesis. We performed ChIP-Chip to identify genes with altered Histone H3 acetylation in primary AML specimens (n=115) and normal CD34+ progenitor cells (n=21). Interestingly, promoters of several members of the peroxiredoxin family (PrdxII and Prdx IV) were identified to be significantly Histone H3 hypoacetylated in primary AML blasts. Peroxiredoxins (Prdx) are antioxidant enzymes that regulate the amount of reactive oxygen species in the cell. Prdx scavenge H2O2 and protect cells from oxidative damage to cellular DNA, lipid and proteins. Confirmation by quantitative PCR revealed that the PrdxII promoter was hypoacetylated in AML, and mRNA expression was 10-fold induced in U937 cells upon exposure to the demethylating agent 5-Azadeoxycytidine (Aza). Demethylation of U937 cells by Aza resulted in increased Histone H3 acetylation at the PrdxII promoter. DNA hypermethylation was frequent in primary AML blasts (18/103) but not in control samples (0/40) as assessed by methylation-specific PCR. Hypoacetylation of Histone H3 and DNA hypermethylation was associated with repression of PrdxII mRNA and protein levels in primary AML samples. Decreased PrdxII protein expression as assessed by immunohistochemistry in a tissue microarray was associated with a poor prognosis in AML patients. On the functional level, PrdxII inhibited the growth of hematopoietic progenitor cells in colony assays and negatively influenced receptor tyrosine kinase signaling by inhibition of ERK and AKT activation upon overexpression. Conversely, PrdxII knock-down by shRNA in myeloid progenitor cell lines and in murine primary bone marrow cells led to enhanced growth and tyrosine kinase signaling. A decrease in PrdxII levels was associated with an increase in reactive oxygen species and enhanced phosphorylation of STAT5, AKT and ERK. Taken together, these findings suggest that chromatin modifications in AML suppress expression of PrdxII. Our data also revealed that PrdxII is a novel putative tumor suppressor in AML.

The Histone Deacetylase Inhibitor LAQ824 Maintains Normal Hematopoietic Progenitor cells (HPC) Associated with Induction of Notch Target Genes and Does Not Eliminate Leukemic HPC in Vitro.

Introduction: Histone deacetylase inhibitors (DACi) have shown promising antileukemic activity by overcoming the differentiation block and inducing apoptosis in AML blasts. Recent data demonstrating enhanced maintenance and functional capacity of normal, but also leukemic hematopoietic progenitor cells (HPC) by the selective class I DACi valproic acid (VPA) have raised concerns about VPA in AML therapy. As more potent pan-DACi have entered clinical trials, we analysed the impact of the hydroxamic acid LAQ824 on phenotype and function of normal and leukemic CD34+ HPC and studied LAQ824- induced gene expression in the most primitive CD34+CD38- population of normal HPC.Methods: Differentiation and proliferation of CD34+ cells of bone marrow of healthy donors and peripheral blood samples of newly diagnosed AML patients were evaluated after one week of culture in presence of SCF, FLT3 ligand, TPO, IL-3 +/− LAQ824. The effect of LAQ824 on gene expression profiles in normal CD34+CD38− cells was assessed in three independent cell samples following incubation with cytokines +/− LAQ824 for 48 hours using Affymetrix GeneChip Human Genome U133 Plus 2.0 and Gene Spring Software. Serial replating of murine Sca1+Lin- HPC was performed in the presence of SCF, G-CSF, GM-CSF, IL-3, IL-6 +/− LAQ824.Results: Treatment of murine Sca1+Lin- HPC with LAQ824 (10 nM) significantly augmented colony numbers (p<0.01; n=3), and supported colony growth after four cycles of replating whereas no colonies developed in its absence beyond the second plating indicating preservation of functionally active multipotent progenitor cells. LAQ824 (10–20 nM) mediated acetylation of histone H3 in human normal and leukemic HPC. In normal HPC, LAQ824 (0–20 nM) lead to a dose-dependent increase in the proportion of CD34+ cells (20% w/o LAQ824 vs. 36% with LAQ824 20nM, p=0.07) and a significant reduction of CD14+ monocytes (18% vs. 3%, p= 0.02; n=3). The total number of CD34+ cells remained stable up to 10 nM and decreased at 20 nM. Gene expression analysis showed, that LAQ824 (20 nM) lead to an at least 3-fold up-regulation of 221 genes in all three HPC samples tested including HDAC11 and the cell cycle inhibitor p21waf1/cip1 known to be induced by most DACi in HPC. We identified several members of the notch pathway such as mastermind-like protein 2 (MAML2, a component of the active notch transcriptional complex) and notch target genes including the transcription factors HES1, HEY1 and HOXA10 and confirmed increase of protein levels by Western blotting. Reduced gene expression of mini-chromosome-maintenance (MCM) protein family members was observed which - in addition to up-regulation of p21 - has previously been associated with notch-mediated cell cycle arrest. To compare the effect of LAQ824 (20 nM) with VPA (150 ng/ml) on leukemic HPC, cells were cultured for one week with or w/o DACi. Of note, LAQ824 resulted in a 0.8-fold reduction of CD34+ leukemic HPC, while VPA expanded this population 2.2-fold compared with cytokine-treated controls (p=0.03; n=12). CFU numbers growing from CD34+ leukemic HPC in presence of LAQ824 did not differ significantly from controls (n=9). Conclusion: LAQ824 seems to diminish, but not eliminate normal as well as leukemic HPC as determined by phenotypic and functional in vitro analyses. Our gene expression analysis suggested an association with coactivator and target genes of the notch pathway and cell cycle arrest-inducing genes. In contrast to VPA, LAQ824 does not seem to support growth of leukemic HPC which may contribute to its more potent antileukemic effect.

SHP-2 Knockdown Results in Profound Inhibition of Human CD34+ Hematopoietic Stem/Progenitor Cell Survival, Proliferation and Differentiation in Response to Growth Factor Stimulation

SHP-2 (ptpn11), a Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase, is expressed at high levels in hematopoietic cells and regulates downstream signaling from growth factor (GF) receptors. SHP-2 has been shown to play an important role in murine hematopoiesis. Moreover, several SHP-2 activating mutations have been identified in myeloid malignancies and there is interest in the development of SHP-2 inhibitors for cancer treatment. On the other hand previous report suggested that SHP-2 inhibition was associated with enhanced GF responsiveness in human hematopoietic cell lines. However the role of SHP-2 signaling in normal human hematopoietic stem and progenitor cell growth has not been studied. Here we investigated the function of SHP-2 in normal human hematopoiesis by inhibiting SHP-2 expression in cord blood (CB) CD34+ cells with stable SHP-2 shRNA expression. We transduced CB CD34+ cells with lentivirus vectors coexpressing SHP-2 specific shRNAs (Si-1 or Si-2) or a control shRNA (Ctrl) and RFP and selected RFP expressing CD34+ cells by flow cytometry sorting. We observed >80% inhibition of SHP-2 expression by Western blotting in Si-1 or Si-2 shRNA transduced cells compared with Ctrl shRNA transduced cells. We observed that culture with increasing concentrations of GF was associated with markedly reduced GF-induced stimulation of proliferation of SHP-2-knockdown CD34+ cells compared to controls. In addition we observed significantly increased apoptosis of SHP-2-knockdown CD34+ cells cultured under low and high GF conditions compared to controls, but little increase in apoptosis in GF-deprived cells, indicating markedly reduced response of SHP-2-knockdown cells to GF-mediated promotion of cell survival. SHP-2-knockdown CD34+ cells also demonstrated significantly reduced expansion in cell numbers following culture in high GF conditions compared with controls (115.3, 25.5 and 10.4 fold expansion for Ctrl, Si-1 and Si-2 at day 7). Analysis of the nature of cells generated in GF culture showed significantly reduced generation of both myeloid (CD33+, CD11b+ and CD14+) and erythroid cells from SHP-2-knockdown CD34+ cells compared with controls, with relatively greater inhibition of myeloid compared with erythroid differentiation. On the other hand CD34+ cell numbers were retained at levels similar to controls after culture. We also observed significantly reduced cell expansion and differentiation and higher apoptotic rates of SHP-2-knockdown cells cultured under either myeloid promoting (IL-3+SCF+G-CSF+GM-CSF) or erythroid promoting (SCF+EPO) GF conditions. SHP-2-knockdown cells demonstrated reduced activation of MAPK and STAT5 but not Akt on Western blotting that was associated with reduced MCL-1 expression, consistent with their reduced GF mediated proliferation and survival. Expression of the transcription factors SCL1, GATA-1, NF-E2 and FOG-1 was increased in SHP-2 knockdown CD34+ cells compared to controls, consistent with the relatively higher retention of CD34+ and erythroid cells compared with myeloid cells after culture. In conclusion, we show that SHP-2 knockdown in human CD34+ cells results in markedly decreased responsiveness to GF stimulation with significantly increased apoptosis, markedly diminished proliferation and reduced generation of differentiated cells during GF culture. A relative retention of the CD34+ cell population was seen despite increased apoptosis, which may be the result of reduced cell turnover and altered transcription factor expression in SHP-2-knockdown cells, and is in contrast to reduced stem cell self-renewal observed following SHP-2 knockdown in murine models. These results indicate a critical role for SHP-2 in GF mediated signaling responses in human hematopoietic stem/progenitor cells. These studies also caution that therapeutic SHP-2 inhibition could be associated with significant hematopoietic toxicity.

Treatment of mice with the Ah receptor agonist and human carcinogen dioxin results in altered numbers and function of hematopoietic stem cells

The aryl hydrocarbon receptor (AhR) mediates the carcinogenicity of a family of environmental contaminants, the most potent being 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increased incidence of lymphoma and leukemia in humans is associated with TCDD exposure. Although AhR activation by TCDD has profound effects on the immune system, precise cellular and molecular mechanisms have yet to be determined. These studies tested the hypothesis that alteration of marrow populations following treatment of mice with TCDD is due to an effect on hematopoietic stem cells (HSCs). Treatment with TCDD resulted in an increased number and proliferation of bone marrow (BM) populations enriched for HSCs. There was a time-dependent decrease in B-lineage cells with a concomitant increase in myeloid populations. The decrease in the B-cell lineage colony-forming unit-preB progenitors along with a transient increase in myeloid progenitors were consistent with a skewing of lineage development from lymphoid to myeloid populations. However, HSCs from TCDD-treated mice exhibited diminished capacity to reconstitute and home to marrow of irradiated recipients. AhR messenger RNA was expressed in progenitor subsets but is downregulated during HSC proliferation. This result was consistent with the lack of response following the exposure of 5-fluorouracil-treated mice to TCDD. The direct exposure of cultured BM cells to TCDD inhibited the growth of immature hematopoietic progenitor cells, but not more mature lineage-restricted progenitors. Overall, these data are consistent with the hypothesis that TCDD, through AhR activation, alters the ability of HSCs to respond appropriately to signals within the marrow microenvironment.

Z3, a novel Jak2 tyrosine kinase small-molecule inhibitor that suppresses Jak2-mediated pathologic cell growth

Jak2 tyrosine kinase is essential for animal development and hyperkinetic Jak2 function has been linked to a host of human diseases. Control of this pathway using Jak2-specific inhibitors would therefore potentially serve as a useful research tool and/or therapeutic agent. Here, we used a high-throughput program called DOCK to predict the ability of 20,000 small molecules to interact with a structural pocket adjacent to the ATP-binding site of murine Jak2. One small molecule, 2-methyl-1-phenyl-4-pyridin-2-yl-2-(2-pyridin-2-ylethyl)butan-1-one (herein designated as Z3), bound to Jak2 with a favorable energy score. Z3 inhibited Jak2-V617F and Jak2-WT autophosphorylation in a dose-dependent manner but was not cytotoxic to cells at concentrations that inhibited kinase activity. Z3 selectively inhibited Jak2 kinase function with no effect on Tyk2 or c-Src kinase function. Z3 significantly inhibited proliferation of the Jak2-V617F-expressing, human erythroleukemia cell line, HEL 92.1.7. The Z3-mediated reduction in cell proliferation correlated with reduced Jak2 and STAT3 tyrosine phosphorylation levels as well as marked cell cycle arrest. Finally, Z3 inhibited the growth of hematopoietic progenitor cells isolated from the bone marrow of an essential thrombocythemia patient harboring the Jak2-V617F mutation and a polycythemia vera patient carrying a Jak2-F537I mutation. Collectively, the data suggest that Z3 is a novel specific inhibitor of Jak2 tyrosine kinase.

Cyclic Nucleotide-Regulated Proliferation and Differentiation Vary in Human Hematopoietic Progenitor Cells Derived from Healthy Persons, Tumor Patients, and Chronic Myelocytic Leukemia Patients

Although circulating hematopoietic progenitor cells (HPCs) are frequently used in therapeutic approaches, many aspects of their cellular biochemistry are still unclear. In the present study, the effects of cyclic nucleotide-elevating agents on HPC proliferation and differentiation were investigated. HPCs from different sources, including healthy persons, patients with tumors (medulloblastoma, seminoma, or multiple myeloma), and patients with chronic myelocytic leukemia (CML), were compared. HPCs were isolated by standard leukapheresis procedures and analyzed for proliferation and differentiation into the megakaryocytic and granulocytic lineages. HPCs contained high concentrations of cyclic guanosine monophosphate (cGMP)-dependent and cyclic adenosine monophosphate (cAMP)-dependent protein kinases G and A (PKG and PKA, respectively). Whereas PKG was partly down-regulated during culture, the PKA level remained constant. Stimulation of PKG in HPCs isolated from healthy donors or tumor patients resulted in a biphasic reaction: low cGMP concentrations inhibited proliferation and stimulated differentiation into megakaryocytes, whereas high concentrations revealed the opposite effect. In contrast, differentiation into granulocytes was inhibited in a concentration-dependent manner. Stimulation of PKA inhibited HPC differentiation; however, HPC proliferation was inhibited in controls and stimulated in HPCs from tumor patients. HPCs isolated from CML patients showed a nonhomogeneous reaction pattern to both cyclic nucleotides with high variability between the individual donors. We demonstrated the importance of the source of HPCs for the investigation of proliferation and differentiation. Cyclic nucleotide-regulated pathways are clearly involved in HPC proliferation and differentiation. Pharmacological strategies using cyclic nucleotide-elevating substances to influence HPC growth and differentiation in the bone marrow might support current strategies in HPC recovery from the peripheral blood.

Effects of peritoneal lavage fluid from radiation or/and burn injured rats on the growth of hematopoietic progenitor cells

Purpose: To evaluate the effects of peritoneal lavage fluids from radiation injury, burn injury and combined radiation-burn injury on the growth of hematopoietic progenitor cells (HPC).Materials and methods: Rats were divided into four groups: A radiation group (RG), a burn group (BG), a combined radiation-burn group (CRBG) and normal control group (NG). RG and CRBG rats were irradiated with 12 Gy, and burns of 30% total body surface area were generated in group BG and group CRBG. Peritoneal lavage fluids were collected and tested for their effects on the growth of erythrocyte progenitor cells or granulocyte-macrophage progenitor cells of BALB/c mice in vitro.Results: The numbers of colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E) and colony-forming units-granulocyte-macrophage (CFU-GM) formed after treatment with lavage fluids from BG or CRBG were significantly higher than those from NG. However, fewer CFU-E, BFU-E or CFU-GM colonies were found after treatment with lavage fluid from the RG. In lavage fluid from BG and CRBG, the concentration of interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor α (TNFα) was increased in comparison to NG and RG. Treatment with these cytokines had similar promoting effects on the growth of hematopoietic colonies and neutralizing antibodies inhibited these effects significantly.Conclusions: Burns increase the responsiveness of the system and help the proliferation of hematipoietic progenitor cells, while radiation decreases all these responses relative to both the controls and the burn plus radiation group.

Hematopoietic Progenitor Cells Mobilize to the Site of Injury After Trauma and Hemorrhagic Shock in Rats

Trauma and hemorrhagic shock (T/HS) has been demonstrated to result in bone marrow (BM) suppression and the release of hematopoietic progenitor cells (HPC) into the peripheral blood in both human beings and experimental animals. HPC have also been identified in numerous end organs after T/HS and the ongoing loss of progenitor cells from the BM may play a role in posttraumatic BM suppression. We investigated the hypothesis that HPC will specifically migrate to sites of tissue trauma and that this process is exacerbated by hemorrhagic shock (HS). Sprague-Dawley rats (250-400 g) sustaining a unilateral lung contusion (LC) secondary to a blast wave of a percussive nail gun, were subjected to either HS (MAP 40-45 mm Hg for 45 minutes) or sham shock (SS). Animals were killed at 3 hours, 3 days, and 7 days after resuscitation and the right and left lungs from each animal were processed separately and the uninjured left lung served as a control for comparison with the contused right lung. BM mononuclear cells from each individual lung and the femurs were isolated and plated (2 x 10) in duplicate for granulocyte-macrophage colony-forming units (CFU-GM), erythroid colony-forming units (CFU-E), and erythroid burst-forming units (BFU-E) colony growth. At 3 hours, LC resulted in a significant increase in progenitor colonies able to be grown from the injured lung compared with from the uninjured lung (CFU-GM: 11 +/- 1 vs. 5 +/- 2, CFU-E: 12 +/- 7 vs. 5 +/- 3, BFU-E: 7 +/- 1 vs. 3 +/- 1 colonies per 10 BM mononuclear cells; all p < 0.05). HS resulted in a significant increase of the number of colonies of all three cell types in both the uninjured and the contused lung (all p < 0.05). At day 3 after HS, BM progenitor growth remained suppressed whereas the number of cells recoverable from the lung returned toward baseline. By day 7, hematopoietic progenitor cell growth in the BM and the number of those cells able to be grown from the lung returned to levels observed in unmanipulated rats. Unilateral LC results in the rapid mobilization of a significant number of HPC from the BM to the site of injury. BM function is maintained under this condition. The addition of HS increases HPC mobilization from the BM and sequestration at the site of injury as well as decreasing BM HPC growth. We postulate that the accumulation of progenitor cells in the injured tissue combined with an alteration of normal BM homing, as exemplified by the decrease in progenitor cells from the lung without restoration of BM function, plays a role in posttraumatic BM suppression. The mechanism of shock-mediated mobilization from the BM and the exact role and fate of these cells at the site of injury requires further investigation.

Differential Effects of G Protein–Coupled Receptors on Hematopoietic Progenitor Cell Growth Depend on their Signaling Capacities

We have shown that CD34(+) hematopoietic progenitor and stem cells (HPCs) consistently express several G protein-coupled receptors (GPCRs): the chemokine receptor CXCR4, the cysteinyl-leukotriene receptor cysLT1, and receptors for sphingosine 1-phosphate (S1P), particularly S1P1. These GPCRs differentially mediate chemotactic, adhesive, and proliferative responses in HPCs. To elucidate the diversity of the responses observed, we compared their signaling capacities in CD34(+) cells. In primary CD34(+) progenitors, the strongest effects on calcium signaling (intracellular calcium fluxes) were mediated by cysLT1. Analyses in CD34(+) cell lines revealed that calcium signaling induced by cysLT1 was only partially inhibited by pertussis toxin (PTX), while responses induced by CXCR4 and S1P receptors were completely blocked. These findings indicate that cysLT1 signals via Gi and Gq proteins, while CXCR4 and also S1P receptors (e.g., S1P1) only induce Gi protein-mediated effects. By analysis of downstream signaling, we could provide further evidence that combined activation of PTX-insensitive (Gq-mediated) and PTX-sensitive (Gi-mediated) pathways by cysLT1 may explain the strong and broad effects of cysteinyl-leukotrienes in early hematopoietic cells, while signaling of CXCR4 and S1P1 solely depends on Gi proteins, resulting in effects mainly restricted to migration and adhesion.

Mad2 is required for optimal hematopoiesis: Mad2 associates with c-Kit in MO7e cells

AbstractMitotic arrest deficiency 2 (Mad2) is a component of mitotic spindle checkpoint proteins and is essential for accurate chromosome segregation. We investigated a role for Mad2 in hematopoiesis using Mad2-haploinsufficient (Mad2+/−) mice. Mad2+/− bone marrow (BM) and spleen manifested decreased absolute numbers and cycling status of immature, but not mature, hematopoietic progenitor cells. Mad2+/− BM granulocyte-macrophage colony-forming units (CFU-GMs) did not manifest synergistic proliferation in response to stem cell factor (SCF) plus GM-CSF. The percentage of annexin V+ cells was higher in Mad2+/− than Mad2+/+c-Kit+lin− BM after culture with SCF and GM-CSF. However, no significant difference in phosphorylation of extracellular signal–related kinase (Erk1/2) at Thr202/Tyr204 and Akt at Ser473 between Mad2+/− and Mad2+/+BM c-Kit+lin− cells was observed. Immunoprecipitation assays performed in human MO7e cells demonstrated physical association of c-Kit with Mad2. Moreover, stimulation with SCF plus GM-CSF led to dissociation of Mad2 from c-Kit. Confocal microscopy demonstrated that Mad2 colocalized with c-Kit in the cytoplasm of MO7e cells. These results suggest that Mad2 is involved in synergistic growth of immature hematopoietic progenitor cells in response to SCF plus GM-CSF, effects that may be mediated via physical association of Mad2 with c-Kit.

Erlotinib Effectively Inhibits JAK2V617F Activity and Polycythemia Vera Cell Growth

JAK2(V617F), a mutant of tyrosine kinase JAK2, is found in most patients with polycythemia vera (PV) and a substantial proportion of patients with idiopathic myelofibrosis or essential thrombocythemia. The JAK2 mutant displays a much increased kinase activity and generates a PV-like phenotype in mouse bone marrow transplant models. This study shows that the anti-cancer drug erlotinib (Tarceva) is a potent inhibitor of JAK2(V617F) activity. In vitro colony culture assays revealed that erlotinib at micro-molar concentrations effectively suppresses the growth and expansion of PV hematopoietic progenitor cells while having little effect on normal cells. Furthermore, JAK2(V617F)-positive cells from PV patients show greater susceptibility to the inhibitor than their negative counterparts. Similar inhibitory effects were found with the JAK2(V617F)-positive human erythroleukemia HEL cell line. These data suggest that erlotinib may be used for treatment of JAK2(V617F)-positive PV and other myeloproliferative disorders.

Effects of Serum From Rats With Combined Radiation-Burn Injury on the Growth of Hematopoietic Progenitor Cells

This study aims to observe the effects of blood serum from rats with radiation injury, burn injury, and combined radiation-burn injury on the growth of hematopoietic progenitor cells and to explore the possible mechanisms. Serum from rats with radiation injury, burn injury, and combined radiation-burn injury were collected at 3 hours, 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours after injury and then was added to the culture medium to see its effect on the growth of hematopoietic progenitor cells (HPCs) at a final protein concentration of 10 microg/mL. Radioimmunoassay and enzyme-linked immunosorbent assay were employed to measure the level of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 in each group, and the effect of TNF-alpha and IL-6 on the growth of HPC was also observed. The number of HPCs colonies formed after addition of the serum from rats with burn or combined radiation-burn injuries was significantly higher than that from normal rats at 3 hours, 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours after injury and reached its peak value at 24 hours after injury. However, fewer HPCs colonies were found after the addition of the serum from irradiated rats. At the same time, the levels of TNF-alpha and IL-6 in the serum of burn group and combined radiation-burn injury group were significantly higher than that of normal group, and much higher than that of the irradiation injury group (p < 0.01). Also, TNF-alpha and IL-6 demonstrated promoting effect on the growth of HPC. Serum from rats with burn injury and combined radiation-burn injury stimulates the growth of HPCs, while serum from irradiated rats shows inhibitory effects on the growth of HPCs. These effects may lie in the different level of TNF-alpha and IL-6 in the serum of each group.

High meningioma 1 (MN1) expression as a predictor for poor outcome in acute myeloid leukemia with normal cytogenetics

AbstractThe translocation t(12;22) involves MN1 and TEL and is rarely found in acute myeloid leukemia (AML). Recently, it has been shown in a mouse model that the fusion protein MN1-TEL can promote growth of primitive hematopoietic progenitor cells (HPCs) and, in cooperation with HOXA9, induce AML. We quantified MN1 expression by real-time reverse transcriptase–polymerase chain reaction (RT-PCR) in 142 adult patients with AML with normal cytogenetics treated uniformly in trial AML-SHG 01/99. AML samples were dichotomized at the median MN1 expression. High MN1 expression was significantly correlated with unmutated NPM1 (P < .001), poor response to the first course of induction treatment (P = .02), a higher relapse rate (P = .03), and shorter relapse-free (P = .002) and overall survivals (P = .03). In multivariate analysis, MN1 expression was an independent prognostic marker (P = .02) in addition to age and Eastern Cooperative Oncology Group (ECOG) performance status. Excluding patients with NPM1mutated/FLT3ITDnegative, high MN1 expression was associated with shorter relapse-free survival (P = .057). MN1 was highly expressed in some patients with acute lymphoblastic but not chronic lymphocytic or myeloid leukemia. MN1 was highly expressed in HPCs compared with differentiated cells and was down-regulated during in vitro differentiation of CD34+ cells, suggesting a functional role in HPCs. In conclusion, our data suggest MN1 overexpression as a new prognostic marker in AML with normal cytogenetics.

High Expression of ENPP2/Autotaxin in Patients with FLT3-ITD Positive Acute Myeloid Leukemia.

The Fms-like tyrosine kinase 3 (FLT-3) receptor tyrosine kinase acts as an important player in controlling the growth and differentiation of haematopoietic progenitor cells. It was shown that activating mutations of FLT-3 are involved in the pathogenesis of acute myeloid leukemia (AML). The most frequent aberration is an internal tandem duplication (ITD) of the juxtamembrane domain coding sequence, which is present in up to 27% of the patients with AML and has been associated with poorer prognosis. To further understand the factors involved, we performed microarray analysis to identify genes specifically upregulated in cases with FLT3-ITD mutations. One gene we identified was ENPP2/Autotaxin (ENPP2/ATX). The ENPP2/ATX protein acts as a lysophospholipase D through generating lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). These two bioactive small lipids have crucial functions in cell migration and proliferation and act via G-protein coupled receptors. ENPP2/ATX has been associated with tumor cell migration in several solid tumors, expression has not been reported in myeloid cells before. The aim of the current study was to investigate the expression pattern of ENPP2/ATX in normal haematopoiesis and leukemia as well as its role in the pathogenesis of AML. Real-Time PCR analysis of 215 primary AML samples from all major cytogenetic risk groups confirmed a significant upregulation in FLT3-ITD patients. High expression was also found in cases with complex karyotype and −5 and −7, but not in cases with good risk cytogenetics. To explore the function of ENPP2/ATX in the haematopoietic system, we stably overexpressed two different splice variants of ENPP2/ATX in several leukemic cell lines. The expression was detected at both, mRNA and protein level by RT-PCR and Western blot analyses, respectively. The lysophospholipase D activity of the protein could be measured by a functional assay using an artificial substrate for ENPP2/ATX. Transwell migration assays showed an increased migration capacity of cells with high expression of ENPP2, which indicates that the overexpressed protein is functional. Based on our results, we suggest that ENPP2/ATX is involved in leukemic cell migration. Our findings point to a close link between ENPP2/ATX and FLT3 in the pathogenesis of AML. Current work focuses on its role in cell survival and prognosis.

Targeting the IRF2 Transcription Factor To Inhibit Leukaemic Cell Growth.

Activating mutations of the Ras genes occur at high frequency in acute myeloid leukemia (AML). We have previously shown that expression of mutant N-ras(N-rasm) in murine hematopoietic stem cells is sufficient to induce a myeloid malignancy that resembles human AML(Mackenzie et al. Blood, 1999, 93, 2043–2056). In a 'humanised' NOD/SCID mouse model N-rasm induced a pre-leukemic condition characterised by myeloid proliferation of human hematopoietic progenitor cells in the bone marrow of recipient mice (Shen et al. Exp. Hematol., 2004, 32: 852–860). Even though Ras usually acts as a dominant transforming oncogene, in primary cells and some cancer cell lines, Ras inhibits cell growth. We have previously shown that ectopic expression of N-rasm in leukemia U937 and K562 cells leads to growth suppression (Passioura et al. Cancer Res. 2005, 65, 797–804). The expression profile induced by N-rasm in these cells included the up-regulation of transcription factor Interferon Regulatory Factor1 (IRF1) and activation of cdk inhibitor p21WAF. IRF1 was previously defined as a tumour suppressor, and as such is a target of oncogenic mutations in AML. Antisense suppression of IRF1 prevented N-rasm induced growth suppression and up-regulation of p21WAF1. These results defined a novel tumour suppressive response to oncogenic N-rasm in leukemia cells. A retroviral cDNA library screen for genes that counteract N-rasm-induced growth suppression identified the gene for the Interferon Regulatory Factor2 (IRF2), and as confirmation of the screen, over-expression of IRF2 in leukemia U937 cells acted to inhibit N-rasm-induced growth suppression (Passioura et al. Oncogene . 2005; 24: 7327–36). IRF2 is known for its oncogenic properties and can antagonise IRF1-mediated tumour suppression. In addition, IRF2 is often up-regulated in primary leukemia samples. Here we show that IRF2 gene suppression using RNA interference acts to suppress the growth of leukemia TF-1 cells bearing N-ras mutation in codon 61 and expressing high levels of IRF1 and IRF2 and low level of p21Waf1. IRF2 down-regulation confirmed at RNA (quantitative RT-PCR) and protein (Western analysis) levels resulted in up-regulation of p21Waf1 and G2/M- rather than G1/S-growth arrest. In addition, increased polyploidisation that results from discoordinated DNA synthesis in mitotically arrested cells, was observed. In addition, IRF2-down-regulation significantly reduced clonogenic growth of the leukemic blasts. Cell growth of normal hematopoietic progenitor cells that express low levels of both IRF1 and IRF2, however, was not affected by IRF2 targeting. IRF2 targeting is currently being examined in primary AML samples in an animal model of AML. We suggest that IRF2 suppression can be used for ex vivo purging of leukemia cells in the autologous stem cell transplantation setting. To the best of our knowledge, specific IRF2 inhibition in cancer cells as a potential therapeutic approach has not been tested to date. IRF2 suppression may prove to be a novel therapeutic target for leukemia therapy.

Altered Expression and Signalling of Bone Morphogenetic Protein Receptor 1A (BMPR-1A) and Enhanced Hematopoietic Progenitor Cell Growth in Long-Term Stromal Culture Assays from Two Patients with a Myeloproliferative Syndrome and Pulmonary Hypertension.

The rare association of pulmonary hypertension and myeloproliferative syndromes (MPS) has been described previously (Garcia-Manero G et al., Am J Hematol 1999; 60(2): 130-5, Dingli D et al., Chest 2001, 120(3): 801-8), but the mechanisms contributing to this potentially hazardous condition await further elucidation. Bone morphogenetic protein receptors (BMPR) modulate the size of the hematopoetic niche (Zhang J et al., Nature 2003, 425(6960): 836-41), with an inhibitory effect on myeloproliferation. In the pulmonary vascular bed, a decreased or deficient expression of BMPR has been shown to result in the proliferation of vascular smooth-muscle cells, asymmetric neointimal hyperplasia in small pulmonary arteries and subsequent pulmonary hypertension (Lane KB et al., Nat Genet 2000, 26(1):81-4; Du L et al, N Engl J Med 2003, 348(6): 500-9). We hypothesized that, in patients suffering from MPS and pulmonary hypertension, changes in the expression of BMPR and subsequent signalling molecules Angiopoietin-1 (Ang-1) and its receptor, TIE-2, may occur not only in the lung, but also in the bone marrow and correspond with enhanced myeloproliferation in vitro.Bone marrow stromal cells were cultured frompatients with pulmonary hypertension and MPS (n=2),patients with MPS and no evidence of pulmonary hypertension (n=3) andhealthy controls (n=3).The cultured cells were subjected to Western Blot analysis for the expression of BMP receptors BMPR-1A and BMPR-2, Angiopoietin-1 and TIE-2. Furthermore, a modified long-term culture initiating cell (LTC-IC) assay was established using the cultured bone marrow stromal cells as layers for autologous and allogeneic progenitor cell assays.The two patients suffering from both, MPS and pulmonary hypertension, showed a diminished expression of BMPR-1A and an enhanced expression of Ang-1 and TIE-2 in cultured stromal cells when compared to patients with MPS alone and to healthy controls. In one of the two patients, a three- to fourfold increase in the number of long-term culture-initiating cells after seeding of CD34-positive cells and of bone marrow mononuclear cells from healthy donors and from the other patients included was observed in modified LTC-IC assays.Our observations argue in favour of changes in BMP receptor expression and signalling with impact not only on pulmonary hypertension as described before, but also on the emergence of a myeloproliferative state.