Apoptosis Of Hematopoietic Progenitor Cells Research Articles

Role of reactive oxygen species in regulating 27-hydroxycholesterol-induced apoptosis of hematopoietic progenitor cells and myeloid cell lines

Oxysterols are oxygenated derivatives of cholesterol that contain an additional hydroxy, epoxide, or ketone group in the sterol nucleus and/or a hydroxyl group in the side chain of the cholesterol molecule. 27-Hydroxycholesterol (27HC) is a side-chain oxysterol that is oxygenated at the 27th carbon atom of cholesterol. The oxysterol (27HC) is produced via oxidation by sterol 27-hydroxylase (CYP27A1) and metabolized via oxysterol 7a-hydroxylase (CYP7B1) for bile acid synthesis in the liver. A previous study has demonstrated that treatment with the alternative Estrogen receptor alpha (ERα) ligand 27HC induces ERα-dependent hematopoietic stem cell (HSC) mobilization. In addition, Cyp27a1-deficient mice demonstrate significantly reduced 27HC levels and HSC mobilization. Here, we report that exogenous 27HC treatment leads to a substantial reduction in the hematopoietic stem and progenitor cell (HSPC) population owing to significantly increased reactive oxygen species (ROS) levels and apoptosis in the bone marrow (BM). However, 27HC does not influence the population of mature hematopoietic cells in the BM. Furthermore, exogenous 27HC treatment suppresses cell growth and promotes ROS production and apoptosis in leukemic cells. Moreover, acute myeloid leukemia (AML) patients with high CYP7B1 expression (expected to have inhibition of 27HC) had significantly shorter survival than those with low CYP7B1 expression (expected to have an elevation of 27HC). Single-cell RNA-sequencing (scRNA seq) analysis revealed that the expression of CYP7B1 was significantly increased in AML patients. Thus, our study suggests that 27HC may serve as a potent agent for regulating pools of HSPCs and may have an application as a novel therapeutic target for hematological malignancies. Collectively, pharmacological inhibition of CYP7B1 (expected to have an elevation of 27HC) would potentially have fewer long-term hematological side effects, particularly when used in combination with chemotherapy or radiation for the treatment of leukemia patients.

Dysregulated T Cell Response in Bone Marrow Immune Micro-Environment of Patients with Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation

Background: Poor graft function (PGF), a kind of bone marrow (BM) failure syndrome, is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Nevertheless, the exact mechanisms underlying PGF remain unclear. The BM immune micro-environment is considered to be involved in the regulation of murine hematopoiesis. Dysregulated T cell response was found to suppress proliferation and induce apoptosis of hematopoietic progenitor cells in patients with aplastic anemia. Therefore, we conducted a study to analyze the alteration of T cell subpopulations in BM micro-environment of allotransplant patients.Aims: To compare the cellular compositions and function of T cells in BM micro-environment between patients with PGF and good graft function (GGF) after allo-HSCT in Peking University Institute of Hematology.Methods: Using a prospective nested case-control study, the active phenotype and memory phenotype of CD4+ T cells and CD8+ T cells in BM were analyzed by flow cytometry in 12 patients with PGF, 36 matched patients with GGF after allo-HSCT, and 15 healthy donors (HDs). Furthermore, the cytokine secretion function of CD4+ T cells and CD8+ T cells were evaluated after simulation and the level of eight Th1 and Th2 cytokines in BM plasma were detection by cytometric beads assay.Results: The demographic and clinical characteristics were similar between allo-HSCT patients with PGF and those with GGF. Although the PGF patients presented a significant lymphopenia, a notable increased percentage of activated CD8+ T cells was detected in the BM of PGF patients when compared to that in GGF patients (61.7% versus 35.0%, P =.02). Moreover, the in vitro cytokine stimulated tests demonstrated a significant higher proportion of Tc1 in PGF patients (46.1% versus 20.3% versus 28.4%, P <.005), an elevated percentage of Th1 in PGF compared with HDs (38.5% versus 21.7%, P <.005), a higher percentage of Th2 (4.5% versus 2.1% versus 2.3%, P <.005) and a dramatically decreased percentage of Tc2 in PGF (0.6% versus 2.0% versus 2.0%, P <.0001). Therefore, a significant elevation in the ratio of Th1/Th2 (19.73 versus 7.39 versus 6.91, P <.0001) and Tc1/Tc2 (67.25 versus 10.07 versus 14.57, P <.005) were observed in PGF when compared with those in GGF and HDs. The changes of IFN-gama and IL-4 levels in BM plasma detected by cytometric beads assay were in accordance with the intracellular cytokine results analyzed by flow cytometry.Summary/Conclusion: Both the in vitro intracellular cytokine testing after stimulation and the BM plasma cytokine detection provides evidence that CD4+ and CD8+ T cells were polarized towards a type-1 cytokine response in patients with PGF, suggesting that the dysfunction of T cell response in BM immune micro-environment may hamper the hematopoietic recovery after allo-HSCT.Acknowledgment: Supported by the National Natural Science Foundation of China (grant nos. 81370638&81230013), and the Beijing Municipal Science and Technology Program (grant nos. Z141100000214011& Z151100004015164& Z151100001615020). DisclosuresNo relevant conflicts of interest to declare.

Excessive TNF-α Production Results from Reduction of c-Fos Via Aberrant Expression of Mir-34a in Neutrophils from Myelodysplastic Syndrome Patients

The molecular basis of ineffective hematopoiesis in myelodysplastic syndromes (MDS) has yet to be defined. Elevation of tumor necrosis factor (TNF)-α in bone marrow plasma has been considered to be a cause of apoptosis of hematopoietic progenitor cells, and this could result in ineffective hematopoiesis in MDS. However, the mechanism of TNF-α elevation has not been elucidated. We recently found aberrant regulation of c-fos mRNA stability in neutrophils isolated from MDS patients (Feng et al, PLoS ONE, 2013). Since the expression levels of RNA-binding proteins that regulated c-fos mRNA stability were not altered in the majority of the patients and no mutations were detected in c-fos mRNA, microRNAs were possibly involved in the insufficient stabilization of c-fos mRNA.In this study, we chose 20 microRNAs targeting c-fos according to four different databases and compared their expression levels in neutrophils between MDS patients and healthy volunteers. Among them, miR-34a and miR-155 were significantly increased in MDS (p<0.05, p<0.05). Aberrantly high expressions of miR-34a and miR-155, which were defined as greater levels than the average in the healthy cells plus 2 standard deviations, were respectively detected in 14 and 13 out of 23 patients with RCUD, RCMD, and RAEB-1. We next examined the effects of the microRNA overexpression on c-fos expression. When miR-34a and miR-155 were introduced into HL60 cells by electroporation to increase their levels to 100-fold or higher than those in the control cells, c-fos protein levels decreased to 35.0 ± 10.9% and 47.8 ± 6.8% of that in the control cells, respectively, while mRNA was not altered. In neutrophils from the patients, c-fos protein but not mRNA was significantly decreased in 13 out of 17 patients tested. The c-fos protein levels were inversely correlated with miR-34a levels (r = -0.618, p<0.05) but not with those of miR-155 (r = -0.135), suggesting that c-fos protein levels were more affected by miR-34a than miR-155.Recently, it was reported that c-fos physically interacted with nuclear factor κB (NF-κB) p65 to inhibit transcription of TNF-α in response to pro-inflammatory cytokines such as lipopolysaccharide (LPS) in monocytic cells (Koga et al, Immunity, 2009). Therefore, we hypothesized that neutrophils with reduced c-fos would overproduce TNF-α in response to inflammatory stimuli in MDS. To test our hypothesis, siRNA for c-fos was introduced into HL60 cells that were differentiated toward a neutrophil-like phenotype by 1.25% DMSO. The treatment with siRNA decreased c-fos protein levels to 58.8 ± 19.6-fold of that in the control cells. When stimulated with 1 μM LPS for 3 hours, the increase rates of TNF-α mRNA were greater in c-fos siRNA-treated cells (41.2 ± 25.7-fold, p<0.05) than in the control cells (6.1 ± 2.3-fold). Chromatin immunoprecipitation assay demonstrated that LPS increased the binding of NF-κB p65 protein to the promoter region of TNF-α DNA by 2.7 ± 1.0-fold in the control cells. In c-fos siRNA-treated cells, the amounts of NF-κB p65 bound to the TNF-α DNA promoter without stimulation were 2.1 ± 0.3-fold greater than those in the unstimulated control cells, which was further increased to 5.6±0.3-fold by stimulation with LPS (p<0.05 compared to LPS-stimulated controls). These data suggested that neutrophils, as well as monocytic cells, transcribed greater amounts of TNF-α when c-fos was decreased. Neutrophils from patients with similar c-fos levels to those in the controls (Group A, n = 5), from the patients with reduced c-fos (Group B, n = 5), and from healthy controls (n = 8) were cultured in the presence of 1 μM LPS for 3 hours. TNF-α mRNA in control neutrophils was elevated 14.9 ± 3.2-fold. The increase in Group A was 10.1 ± 4.5-fold with no significant difference from the controls, while that in Group B was greater (31.5 ± 15.1-fold, p<0.05). Although neutrophils from Group A secreted similar amounts of TNF-α into the culture medium (143.5 ± 65.7 pg/mL) to the controls (150.6 ± 91.5 pg/mL), Group B produced significantly greater amounts of TNF-α (735.4 ± 65.7 pg/mL, p<0.05 vs. controls, p<0.05 vs. Group A). Thus, our data suggest that the reduction of c-fos resulting from aberrant expression of microRNAs contributes to overproduction of TNF-α under inflammatory stimuli in MDS. DisclosuresNo relevant conflicts of interest to declare.

Comparison Of Lymphocytic Immune Profiles Of Aplastic Anemia and Hypocellular Myelodysplastic Syndrome

BackgroundAplastic anemia (AA) and hypocellular myelodysplastic syndrome (MDS) are two common acquired bone marrow failure diseases. AA is mostly an acquired bone marrow disease caused by cellular and humoral mediated immune attack of hematopoietic stem cells (HSC) due to dysregulation of lymphocytic system, which leads to hematopoietic progenitor cell apoptosis and bone marrow failure. MDS is a group of heterogeneous acquired clonal HSC disorders with ineffective hematopoiesis. Approximately 10% to 20% of MDS manifests a reduced bone marrow cellularity, which comprises hypocellular MDS. There is increasing experimental and clinical indication that an immune-mediated damage to hematopoietic HSCs and changes in the hematopoiesis-supporting microenvironment contribute to the pathogenesis of hypocellular MDS. Because of the similarity of their bone marrow manifestation, hypocellular MDS and AA are often hard to distinguish. Mounting evidence indicates that abnormal activation of cytotoxic T cells plays a crucial role in the pathophysiology of these diseases. One study showed that AA patients have an abnormally activated subpopulation of CD4+ helper cells and a decreased number and function of T regulatory cells in the bone marrow. GVHD mouse models further demonstrated that self-reactive T cells were capable of recognizing non-polymorphic tissue or commensally-derived antigens. Recent literature suggests that immune dysregulation plays a major role in pathogenesis of acquired bone marrow failure disease. However immune profiles of these two diseases have not been thoroughly studied, specially the role of B lymphocyte population. Our study aims to find lymphocytic surface marker expression patterns of hypocellular MDS and AA in both immature cell and lymphocyte populations. MethodsThis retrospective study analyzed flow cytometry lymphocytic antigen expression profiles from patients diagnosed as AA and hypocellular MDS as per standard criteria. A total of 31 AA and 26 hypocellular MDS patient cases were recruited. The bone marrow aspirate/biopsy data, bone marrow aspiration flow cytometry reports, and Complete Blood Counts (CBC)s from individual patients were analyzed. Using side scatter (SSC) vs. CD45 gating flow cytometry panels, we identified immature cell population (SSClow/CD45low) and lymphocyte population (SSClow/CD45high). We then quantitatively analyzed the expression patterns of 33 cluster differentiation (CD) molecules on individual sample. Finally, we compared the CD expression patterns between AA and hypocellular MDS in both cell populations respectively. ResultsCD19 expression was significantly higher in AA than in hypocellular MDS in both SSClow/CD45low cell population (P=0.001) and SSClow/CD45high cell population (P=0.003). Hypocellular MDS contains significantly higher CD34high cells than AA in SSClow/CD45low populations (mean:28.5% vs 8.5%; range; 1% to 94% vs 2% to 27%; P=0.04). However, patients with both diseases similarly contains very few CD34high cells in SSClow/CD45high cell population (mean: 0.6% vs 2.6%; range: 0.0% to 2% vs 0.0% to 32%; P=0.99). Conclusion1. In AA, B cells are highly proliferative in both immature stage and mature stage. This data indicates that B cells which may play a unique role in AA pathogenesis but not in hypocellular MDS.2. In both AA and hypocellular MDS, the majority of lymphocyte population are mature cells. This data suggests that the pathogeneses of both diseases caused by a persistently dysregulated immune microenvironment, not by an acute insult.CD19 expression pattern may be a useful marker to distinguish AA and hypocellular MDS. Disclosures:No relevant conflicts of interest to declare.

WT1 regulates murine hematopoiesis via maintenance of VEGF isoform ratio

AbstractMutations in the Wilms tumor suppressor 1 (WT1) gene are as frequent in acute myeloid leukemia (AML) as in nephroblastma and predict poor prognosis. However, the role of WT1 in hematopoiesis remains unclear. We show that Wt1-deficient mouse embryonic stem cells exhibit reduced hematopoietic potential caused by vascular endothelial growth factor A (Vegf-a)–dependent apoptosis of hematopoietic progenitor cells associated with overproduction of the Vegf-a120 isoform. We demonstrate that Wt1 promotes exon inclusion using a Vegf-a minigene-based splicing assay. These data identify a critical role for Wt1 in hematopoiesis and Vegf-a as a cellular RNA whose splicing is potentially regulated by Wt1. The correction of Wt1 deficiency by treatment with exogenous Vegf-a protein indicates that the Wt1/Vegf-a axis is a molecular pathway that could be exploited for the management/treatment of poor prognosis AMLs.

Adenylate Kinase 2 Regulates Zebrafish Primitive and Definitive Hematopoiesis

AbstractAbstract 1208 Introduction:The adenylate kinase (AK) gene family consists of 7 different members that contribute to energy cell metabolism by converting ATP + AMP to 2 molecules of ADP. AKs are critical players in ensuring cellular energy homeostasis in all tissues and are generally involved in a broad range of cellular functions. Among AKs, AK2 has unique features such as its location in the mitochondrial intermembrane space and critical role in human lymphopoiesis and granulopoiesis. Indeed, mutations of the AK2 gene cause reticular dysgenesis (RD), an autosomal recessive form of severe combined immunodeficiency (SCID) characterized by an early differentiation arrest in the granulocyte lineage and impaired lymphoid maturation. The mechanisms underlying the pathophysiology of RD remain unclear. The phenotype of AK2 deficient animals has not been reported in the literature, but murine lines carrying homozygous inactivating retroviral insertions are embryonically lethal (our personal observations). Objectives:To study the role of AK2 in hematopoietic system development and define the effects of AK2 deficiency, we set out to generate a zebrafish model of RD. Methods:We injected zebrafish embryos with morpholino oligomers specific for the two zebrafish AK2 isoforms and analyzed the serial expression pattern of several hematopoietic markers in developing AK2 morphants. To confirm our observations in AK2 knockdown embryos, we screened a zebrafish DNA library of ENU-induced mutations and recovered a mutant fish line carrying a T371C/L124P missense mutation within the exon 4 of AK2 gene that is predicted to be deleterious for protein stability and function. Results:The downregulation of zebrafish AK2 expression phenocopied the human disease and resulted in strong reduction of developing lymphocytes. In addition, in situ hybridization for GATA1, alpha-globin 1, L-plastin and Odianisidine staining indicated that erythroid development was affected in AK2 morphants during primitive hematopoiesis, while myeloid development was conserved. Furthermore, in situ hybridization studies of the expression of markers of zebrafish definitive hematopoiesis showed abnormalities distributed among all hematopoietic lineages suggesting a broad role of AK2 in zebrafish hematopoiesis. Importantly, the ENU-induced Ak2 mutant recapitulated all the primitive and definitive hematopoietic phenotypes observed in AK2 morphants. Finally, preliminary data suggest that AK2 deficiency (both in morphant and mutant embryos) induces an increased level of reactive oxygen species (ROS) triggering oxidative stress and consequent apoptosis in hematopoietic progenitor cells. Conclusions:Our data provide new insights into the AK2 function and indicate that zebrafish represents a good model for studying the molecular mechanisms involved in RD. To date, our mutant line represents the first example of animal model of this rare and unique human disease. Disclosures:No relevant conflicts of interest to declare.

Activation of Wnt/β-Catenin Protein Signaling Induces Mitochondria-mediated Apoptosis in Hematopoietic Progenitor Cells

The canonical Wnt/β-catenin signaling is activated during development, tumorigenesis, and in adult homeostasis, yet its role in maintenance of hematopoietic stem/progenitor cells is not firmly established. Here, we demonstrate that conditional expression of an active form of β-catenin in vivo induces a marked increase in the frequency of apoptosis in hematopoietic stem/progenitor cells (HSCs/HPCs). Activation of Wnt/β-catenin signaling in HPCs in vitro elevates the activity of caspases 3 and 9 and leads to a loss of mitochondrial membrane potential (ΔΨ(m)), indicating that it induces the intrinsic mitochondrial apoptotic pathway. In vivo, expression of activated β-catenin in HPCs is associated with down-regulation of Bcl2 and expression of Casp3. Bone marrow transplantation assays reveal that enhanced cell survival by a Bcl2 transgene re-establishes the reconstitution capacity of HSCs/HPCs that express activated β-catenin. In addition, a Bcl2 transgene prevents exhaustion of these HSCs/HPCs in vivo. Our data suggest that activation of the Wnt/β-catenin pathway contributes to the defective function of HPCs in part by deregulating their survival.

Changes in Gene Expression Profiles in Patients with 5q- Syndrome Caused by Lenalidomide Treatment

Abstract 5023A direct effects of lenalidomide on gene expression in 5q- patients was studied using HumanRef-8 v2 Expression BeadChips (Illumina). Expression profiles of 6 patients (before treatment and at the time of the first erytroid response) and 6 healthy controls were investigated from CD14+ monocytes of peripheral blood. Differentially expressed genes were identified by Significance Analysis of Microarrays (SAM). Simultaneously, selected genes (TNF, JUN, IL1) were monitored in the course of treatment using Real-Time PCR with Taqman Gene Expression Assays.A comparison of gene expression levels before and during lenalidomide treatment revealed 97 differentially expressed genes (FC >2; p<0.05) related to following biological processes: immune response (16 genes), inflammatory response (11 genes), response to bacteria (8 genes), anti-apoptosis (7 genes), regulation of MAP kinase activity (5 genes), oxygen transport (4 genes), and regulation of cell proliferation (11 genes). An overexpression of a number of cytokines (e.g. TNF, IL8, IL1B, CCL3L, CXCL2, and TNFAIP3) was detected in patients before treatment, after lenalidomide administration expression of the majority of the up-regulated cytokine genes decreased to the control baseline level. Detected overproduction of the cytokines in 5q- syndrome may lead to an increased apoptosis of hematopoietic progenitor cells and together with excessive oxidative stress may contribute to the damage the hematopoietic niche. In the same manner, untreated patients showed suppressed expression of two genes (CXCR4, CRTAP) which play an important role in the stem cell niche. After treatment, we detected increased expression of these genes. Both the observations might explain favorable effects of lenalidomide on the bone marrow stroma defect seen in 5q- syndrome. On the other hand, a substantial increase of the ARPC1B gene (an activator and a substrate of Aurora A) expression was detected after lenalidomide treatment. Since overexpression of Aurora A leads to polyploidy and chromosomal instability, ARPC1B might play a role in the disease progression observed in some patients treated with lenalidomide.To conclude, described changes in genes expression may contribute to identification of the pathways affected by lenalidomide and to the explanation of some effects of this drug that have not been fully understood yet.Supported by grants NS/9634 MZCR, UHKT2005 00023736, MSM0021620808 and COST EUGESMA Disclosures:No relevant conflicts of interest to declare.

Slug suppression induces apoptosis via Puma transactivation in rheumatoid arthritis fibroblast-like synoviocytes treated with hydrogen peroxide.

Inadequate apoptosis contributes to synovial hyperplasia in rheumatoid arthritis (RA). Recent study shows that low expression of Puma might be partially responsible for the decreased apoptosis of fibroblast-like synoviocytes (FLS). Slug, a highly conserved zinc finger transcriptional repressor, is known to antagonize apoptosis of hematopoietic progenitor cells by repressing Puma transactivation. In this study, we examined the expression and function of Slug in RA FLS. Slug mRNA expression was measured in the synovial tissue (ST) and FLS obtained from RA and osteoarthritis patients. Slug and Puma mRNA expression in FLS by apoptotic stimuli were measured by real-time PCR analysis. FLS were transfected with control siRNA or Slug siRNA. Apoptosis was quantified by trypan blue exclusion, DNA fragmentation and caspase-3 assay. RA ST expressed higher level of Slug mRNA compared with osteoarthritis ST. Slug was significantly induced by hydrogen peroxide (H2O2) but not by exogenous p53 in RA FLS. Puma induction by H2O2 stimulation was significantly higher in Slug siRNA-transfected FLS compared with control siRNA-transfected FLS. After H2O2 stimulation, viable cell number was significantly lower in Slug siRNA-transfected FLS compared with control siRNA-transfected FLS. Apoptosis enhancing effect of Slug siRNA was further confirmed by ELISA that detects cytoplasmic histone-associated DNA fragments and caspase-3 assay. These data demonstrate that Slug is overexpressed in RA ST and that suppression of Slug gene facilitates apoptosis of FLS by increasing Puma transactivation. Slug may therefore represent a potential therapeutic target in RA.

Stem Cell Factor Prevents Neuronal Cell Apoptosis After Acute Spinal Cord Injury

A rat spinal cord injury (SCI) model and immunohistochemistry were used to examine the levels of expression of stem cell factor and c-kit. In addition, we examined whether intraperitoneal administration of stem cell factor could prevent neural cells apoptosis after acute SCI. To evaluate the antiapoptotic effect of stem cell factor after SCI. It is well known that the mode of delayed neuronal and glial cell death after SCI is apoptosis. Inhibition of apoptosis might thus promote neurologic improvement after SCI. Stem cell factor and its receptor c-kit exhibit pleiotropic effects in early hematopoiesis, and are also known to prevent hematopoietic progenitor cell apoptosis. Stem cell factor has recently been reported to be a survival factor for neural stem cells in vitro. We examined the levels of expression of stem cell factor and c-kit in normal and injured rat spinal cord. In addition, we examined whether stem cell factor prevents neural cell apoptosis after acute SCI. We examined the expression of stem cell factor and c-kit in spinal cord after injury by quantitative RT-PCR and immunohistochemistry. Antiapoptotic effects of stem cell factor were examined using rats with SCI that received stem cell factor intraperitoneally, and were examined immunohistochemically with anticleaved PARP antibody and antiactive caspase-3 antibody between 1 and 3 days after injury. Upregulation of stem cell factor and c-kit expression occured after SCI. We also found that neurons express stem cell factor, and neurons and oligodendrocytes express c-kit after SCI. In addition, intraperitoneal administration of stem cell factor prevented spinal neural cells apoptosis after injury. These findings suggest the possibility that stem cell factor, a hematopoietic cytokine, could be useful as an agent for treatment of SCI.

Granulocyte-Colony Stimulating Factor Inhibits Neuronal Apoptosis in a Rat Model of Diabetic Cerebral Ischemia

Incidence of cerebral vascular disease (CVD) is higher in patients with diabetes mellitus (DM) than that in individuals without DM, and neuronal apoptosis determines the severity of cerebral infarction. However, there is no effective therapy for CVD. Granulocyte-colony stimulating factor (G-CSF), a potent hematopoietic factor, could inhibit apoptosis of hematopoietic progenitor cells. However, its effect on neuronal cells is still unclear. In this study, we investigated the anti-apoptosis properties of G-CSF in neurons following focal cerebral ischemia in diabetic rats. The diabetic condition was generated in rats by intravenous injection of streptozotocin. After 6 weeks, diabetic rats underwent middle cerebral artery occlusion (MCAO) and received subcutaneous administration of G-CSF (50 microg/kg) daily for 7, 14 or 21 days. We analyzed the changes in neurological severity scores, infarct volume, number of apoptotic neurons, and the expression of G-CSF receptor, phosphorylated signal transducer and activator of transcription 3 (pSTAT3), cellular inhibitor of apoptosis protein 2 (cIAP2), Bcl-2, and Bax in the brain tissue. Bax is a pro-apoptotic member of the Bcl-2 protein family. The DM rats treated with G-CSF not only showed the reduced infarct volume and decreased apoptosis cell number, but also presented improved neurological scores. The G-CSF also increased the expression of pSTAT3, Bcl-2, and cIAP2 proteins as well as Bcl-2 mRNA, but inhibited Bax protein expression in the brain. These results indicate that G-CSF partially increases neuronal survival by affecting apoptosis pathways. G-CSF provides a potential treatment for stroke and other neurological dysfunction accompanied by neuronal apoptosis.

Direct and Indirect Effects of Androgens on Survival of Hematopoietic Progenitor Cells In Vitro

Androgens remain a common treatment for certain type of anemia, based upon its myelostimulating effects; however, it has not been established whether androgens affect apoptosis of hematopoietic progenitor cells (HPCs). We investigated the effects of the androgens, such as testosterone, 5β-dihydrotestosterone (5-DHT), and oxymetholone, on apoptosis of normal hematopoietic progenitor cells in vitro. Androgens did not rescue normal bone marrow (BM) CD34+ cells and colony-forming cells (CFCs), other than mature erythroid CFCs, from apoptosis induced by serum- and growth factor deprivation. Oxymetholone did not affect growth factor-mediated survival of normal CD34+ cells or its inhibition by interferon-gamma (IFN-γ). In a standard methylcellulose clonogenic assay, low concentrations of oxymetholone and 5-DHT stimulated the clonal growth of colony-forming unit (CFU)-erythroid, but did not affect growth of CFU-granulocyte/macrophage or burst-forming unit-erythroid. Oxymetholone and 5-DHT stimulated the production of stem cell factor in normal bone marrow stromal cells (BMSCs) via transcriptional regulation. In agreement with this, oxymetholone-treated BMSCs better supported the survival of HPCs. These data indicate that survival-enhancing or growth-stimulatory effects of androgens on hematopoietic progenitor cells are minimal and mostly restricted to mature erythroid progenitors, and its myelostimulating effects could be attributed, at least in part, to the stimulation of production of hematopoietic growth factors in BMSCs.

Direct and Indirect Effects of Oxymetholone on Growth and Apoptosis of Hematopoietic Progenitor Cells In Vitro.

Despite a tarnished reputation, androgens remain a common treatment for aplastic anemia, especially in the Orient. Increased apoptosis of hematopoietic cells is characteristic of aplastic anemia; however, it has not been established whether androgens affect apoptosis of hematopoietic progenitor cells. In most previous studies regarding the in vitro effects of androgens on hematopoietic cells, whole bone marrow cells were used, rather than purified hematopoietic progenitor cells such as CD34+ cells. With these questions in mind, we investigated the direct and indirect effects of oxymetholone and other androgens on apoptosis and growth of normal hematopoietic progenitor cells (HPCs) in vitro. Oxymetholone did not rescue normal BM CD34+ cells and colony-forming cells (CFCs), other than mature erythroid CFCs, from apoptosis induced by growth factor deprivation. Unexpectedly, both testosterone and 5-dihydrotestosterone (5-DHT) at a concentration of 10−5 M, but not oxymetholone, increased the percentage of annexin-positive apoptotic cells (62.2 ± 5.9%, P < 0.05; 61.7 ± 6.4%, P < 0.05, respectively) compared with the controls (52.6 ± 5.6%). The addition of either stromal cell-derived factor-1 (SDF-1) or stem cell factor (SCF) partially relieved the increase in apoptosis induced by 5-DHT, and the addition of both SDF-1 and SCF completely reversed it. Oxymetholone did not rescue CFCs from interferon-gamma (IFN-g)-induced inhibition of clonal growth of BM CD34+ cells in methylcellulose cultures. Furthermore, oxymetholone did not mitigate IFN-g-induced suppression of CD34+ cell survival in the presence of growth factors. In a methylcellulose clonogenic assay, oxymetholone stimulated the clonal growth of colony-forming unit-erythroid at low concentrations, while not affecting colony-forming unit-granulocyte/macrophage or burst-forming unit-erythroid. Oxymetholone did not reverse the IFN-g-induced inhibition of colony formation by CD34+ cells. Interestingly, oxymetholone stimulated the production of SCF and thrombopoietin in normal human bone marrow stromal cells (BMSCs) through transcriptional regulation while inhibiting the production of interleukin-6. In agreement with this, oxymetholone-treated BMSCs better supported the survival and growth of HPCs. These results suggest that oxymetholone exerts most of its myelostimulatory effects via the regulation of cytokine production in BMSCs, rather than by direct action on hematopoietic progenitor cells.

Internal Tandem Duplication of Flt3 (ITD-Flt3) Bypasses Growth Factor Mediated Survivin Expression, while Disruption of Survivin Accelerates Cell Death in Hematopoietic Cells Expressing ITD-Flt3.

The inhibitor of apoptosis protein Survivin is barely detectable in normal adult tissues but is over-expressed in almost all cancers and hematopoietic malignancies and confers resistance to apoptosis and aberrant cell proliferation. We have shown that hematopoietic growth factors induce Survivin expression in normal CD34+ cells in both cell cycle dependent and independent fashion and that oncogenic Ras can enhance Survivin expression independent of growth factor stimulation. In addition, over-expression of Survivin blocks apoptosis of hematopoietic progenitor cells through a p21WAF1/Cip1 (p21) dependent mechanism. However the mechanisms responsible for Survivin overexpression in malignant hematopoietic cells is poorly understood. Flt3 is a type III tyrosine kinase receptor expressed mainly in the primitive hematopoietic compartment. Internal tandem duplication (ITD) mutations of the Flt3 gene that activate the Flt3 tyrosine kinase are frequently found in patients with AML and associated with poor prognosis. Furthermore, their ectopic expression in IL-3 dependent Ba/F3 cells results in IL-3-independent proliferation and a myeloproliferative disease in mice. In order to understand the mechanism by which ITD-Flt3 enhances cell survival, we examined whether ITD-Flt3 can bypass Survivin up-regulation independent of hematopoietic growth factors and if Survivin lies down stream of ITD-Flt3 in prolonging cell survival. Incubation of primary umbilical cord blood CD34+ cells with Flt3 ligand induced Survivin expression, indicating that Survivin lies down stream of Flt3 signaling pathways in CD34+ cells. While Survivin expression was comparable in Ba/F3 cells expressing ITD-Flt3 (W51, W73 and W78 mutants, kindly provided by Dr. DG Gilliland) and wild-type Flt3 when maintained in the presence of IL-3, ITD-Flt3 prevented down-modulation of Survivin expression induced by IL-3 withdrawal even during G0/G1 phase of cell cycle, suggesting that up-regulation of Survivin is not a consequence of cell cycle progression associated with ITD-Flt3 expression. The increase in Survivin by ITD-Flt3 was associated with reduced active caspase-3 and prolonged cell survival coincident with induction of p21 expression and Tyrosine 15 phosphorylation of CDK1 (Cdc2), which have been implicated in anti-apoptosis. These results are consistent with a p21 dependent anti-apoptosis effect of Survivin in primary hematopoietic progenitor cells and suggest the involvement of Survivin-p21 and CDK1 in ITD-Flt3 mediated cell survival. Overexpression of dominant negative T34A Survivin inhibits the enhanced survival induced by ITD-Flt3 in Ba/F3 cells following IL-3 withdrawal, suggesting that the survival effect of ITD-Flt3 is mediated by Survivin. Similar to ITD-Flt3, overexpression of ectopic wild-type Survivin results in enhanced cell survival in Ba/F3 cells but failed to induce cell cycle progression and IL-3-independent proliferation, indicating that Survivin alone is not sufficient for induction of growth factor independent proliferation and the cell cycle enhancing effect of ITD-Flt3. In summary, our findings indicate that ITD-Flt3 up-regulates Survivin expression independent of hematopoietic growth factors and that the ITD-Flt3/Survivin axis provides a survival advantage in transformed hematopoietic cells. Disruption of these pathways may increase apoptosis of malignant hematopoietic cells and provide therapeutic benefit.

Fas-mediated apoptosis is important in regulating cell replication and death in trisomy 8 hematopoietic cells but not in cells with other cytogenetic abnormalities

Increased apoptosis of hematopoietic progenitor cells has been implicated in the pathophysiology of cytopenias associated with myelodysplastic syndromes (MDSs), and inhibition by immunosuppression may account for the success of this treatment in some patients. We examined bone marrow and peripheral blood of 25 patients with chromosomal abnormalities associated with MDS (monosomy 7, trisomy 8, and 5q-) for evidence of apoptosis. When fresh bone marrow was examined, the number of apoptotic and Fas-expressing CD34 cells was increased in patients with trisomy 8, but decreased in monosomy 7, as compared with healthy control donor marrow. Fas expression was increased in the trisomy 8 cells and decreased in the monosomy 7 cells when compared with normal cells from the same patient. Trisomy 8 cells were more likely to express activated caspase-3 than were normal cells. For bone marrow cells cultured with Fas agonist or Fas antagonist, the percentage of cells with trisomy 8 was significantly decreased in most cases after Fas receptor triggering and increased by Fas ligand (Fas-L) antagonist (P < 0.01), suggesting increased Fas susceptibility of cells with trisomy 8. No such changes were seen in cultures of cells with 5q- or monosomy 7. Fas antagonist facilitated the expansion of cells with trisomy 8 only. Cells with trisomy 8 appear to be more susceptible to Fas-mediated apoptosis. Clinical data demonstrating the responsiveness of some patients with trisomy 8 to anti-thymocyte globulin (ATG) and cyclosporine (CsA) would favor an active role of the immune system in this syndrome.

Tumor necrosis factor-alpha levels in long-term marrow cultures from patients with aplastic anemia: modulation by granulocyte-macrophage colony-stimulating factor.

We have previously shown that the levels of hematopoietic progenitors in long-term marrow cultures (LTMC) from patients with aplastic anemia (AA) are drastically reduced, as compared to normal LTMC. We have also reported that when LTMC from AA patients are supplemented with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) there is an increase in colony-forming cell (CFC) levels. However, such a stimulation is only transient and it is followed by an inhibition in CFC growth. Based on these observations, in the present study we have tested the hypothesis that the levels of tumor necrosis factor-alpha (TNF-alpha), an inhibitor of hematopoiesis, are increased in AA LTMC and that such levels are further increased after rhGM-CSF has been added to the cultures for several weeks. Accordingly, we have determined the levels of TNF-alpha in the supernatant of LTMC established from normal (n = 8) and AA (n = 6) bone marrow and in AA LTMC supplemented with rhGM-CSF (n = 6). At the time of culture initiation, TNF-alpha levels were below detection in all the samples analyzed. After 5 weeks of culture, TNF-alpha levels in normal LTMC were very low, with a median of 7.3 pg/mL. In contrast, AA LTMC contained higher levels of TNF-alpha (median of 49.6 pg/mL). In keeping with our hypothesis, addition of rhGM-CSF to AA LTMC resulted in a significant further increase of TNF-alpha levels (median of 135.4 pg/mL). Our results demonstrate an inverse correlation between reduced hematopoiesis in AA LTMC and increased levels of TNF-alpha in this culture system. Based on the results presented here, together with previous reports indicating that TNF-alpha is a potent inducer of apoptosis in hematopoietic progenitor cells, it seems reasonable to suggest that TNF-alpha is implicated in the pathophysiology of AA.

The role of apoptosis in the pathogenesis of the myelodysplastic syndromes.

The paradoxical occurrence of peripheral cytopenias despite a normo/hypercellular marrow in myelodysplastic syndromes (MDS) has been attributed to excessive intramedullary hematopoietic cell apoptosis. It has also been postulated that abrogation of programmed cell death (PCD) may underlie MDS transformation to acute myeloid leukemia (AML). Despite overwhelming evidence for a role of aberrant apoptosis in myelodysplasia, the molecular mechanisms responsible for such changes have not been elucidated. This paper summarizes current evidence implicating a role for altered PCD in MDS and outlines potential cellular mechanisms whereby hematopoietic progenitor cell apoptosis may be dysregulated.

Apoptosis in megaloblastic anemia occurs during DNA synthesis by a p53-independent, nucleoside-reversible mechanism

Deficiency of folate or vitamin B12 (cobalamin) causes megaloblastic anemia, a disease characterized by pancytopenia due to the excessive apoptosis of hematopoietic progenitor cells. Clinical and experimental studies of megaloblastic anemia have demonstrated an impairment of DNA synthesis and repair in hematopoietic cells that is manifested by an increased percentage of cells in the DNA synthesis phase (S phase) of the cell cycle, compared with normal hematopoietic cells. Both folate and cobalamin are required for normal de novo synthesis of thymidylate and purines. However, previous studies of impaired DNA synthesis and repair in megaloblastic anemia have concerned mainly the decreased intracellular levels of thymidylate and its effects on nucleotide pools and misincorporation of uracil into DNA. An in vitro model of folate-deficient erythropoiesis was used to study the relationship between the S-phase accumulation and apoptosis in megaloblastic anemia. The results indicate that folate-deficient erythroblasts accumulate in and undergo apoptosis in the S phase when compared with control erythroblasts. Both the S-phase accumulation and the apoptosis were induced by folate deficiency in erythroblasts fromp53 null mice. The complete reversal of the S-phase accumulation and apoptosis in folate-deficient erythroblasts required the exogenous provision of specific purines or purine nucleosides as well as thymidine. These results indicate that decreased de novo synthesis of purines plays as important a role as decreased de novo synthesis of thymidylate in the pathogenesis of megaloblastic anemia.

Apoptosis in megaloblastic anemia occurs during DNA synthesis by a p53-independent, nucleoside-reversible mechanism

AbstractDeficiency of folate or vitamin B12 (cobalamin) causes megaloblastic anemia, a disease characterized by pancytopenia due to the excessive apoptosis of hematopoietic progenitor cells. Clinical and experimental studies of megaloblastic anemia have demonstrated an impairment of DNA synthesis and repair in hematopoietic cells that is manifested by an increased percentage of cells in the DNA synthesis phase (S phase) of the cell cycle, compared with normal hematopoietic cells. Both folate and cobalamin are required for normal de novo synthesis of thymidylate and purines. However, previous studies of impaired DNA synthesis and repair in megaloblastic anemia have concerned mainly the decreased intracellular levels of thymidylate and its effects on nucleotide pools and misincorporation of uracil into DNA. An in vitro model of folate-deficient erythropoiesis was used to study the relationship between the S-phase accumulation and apoptosis in megaloblastic anemia. The results indicate that folate-deficient erythroblasts accumulate in and undergo apoptosis in the S phase when compared with control erythroblasts. Both the S-phase accumulation and the apoptosis were induced by folate deficiency in erythroblasts fromp53 null mice. The complete reversal of the S-phase accumulation and apoptosis in folate-deficient erythroblasts required the exogenous provision of specific purines or purine nucleosides as well as thymidine. These results indicate that decreased de novo synthesis of purines plays as important a role as decreased de novo synthesis of thymidylate in the pathogenesis of megaloblastic anemia.

Cell cycle status and apoptosis of hematopoietic progenitor cells released into the peripheral blood after taxanes and granulocyte colony-stimulating factor in breast cancer patients.

Paclitaxel and its analogue docetaxel show a significant antitumor activity, particularly evident in breast cancer. Paclitaxel has also been proved to be effective as a peripheral blood progenitor cell (CPC) mobilizing agent. To optimize the use of active, disease-specific drugs as CPC priming, we have evaluated the effects of either paclitaxel or docetaxel both at standard dosages and followed by granulocyte colony-stimulating factor (G-CSF), on circulating CPC release and function in 18 patients with advanced breast cancer who had failed previous anthracycline-based regimens. The reported differences in biological behaviour between bone marrow and blood-derived hematopoietic progenitor cells and the ability of both paclitaxel and docetaxel to induce apoptosis, prompted us to simultaneously evaluate the cell cycle perturbations induced on CD34+ cells. Median CD34+ peaks were 24 microl (range: 10-58) in the paclitaxel-treated patients and 39 microl (range: 17-91), respectively, in the patients who received docetaxel. After paclitaxel, the percentage of CD34+ cells in S-phase was low (bromodeoxyuridine, BrdU, labelling index = 3.4+/-2%) with a concomitant presence of early apoptotic cells (8.1+/-3%). On the contrary, after docetaxel, the percentage of CD34+ cells in S-phase was higher (BrdU labelling index = 14.5+/-4%, p<0.05 vs. paclitaxel), while early apoptotic cells were detected at a similar rate (8. 6+/-3%, p = n.s. vs. paclitaxel). In conclusion, when used at standard dosages, with respect to paclitaxel + G-CSF, docetaxel + G-CSF is a more satisfactory tool to mobilize CPC and to induce them into the cell cycle. These data should be taken into account when combinations of docetaxel with other agents are explored as CPC mobilizing regimens for autografting.