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PubMed2000

Signal transduction in primary cultured human erythroid cells.

Development of erythrocytes is a complex process governed by multiple cytokines. Colony assays have revealed the physiologic importance of these cytokines, although biochemical studies of highly purified human colony-forming unit-erythroid (CFU-E) generated in vitro from CD34+ cells have only recently begun. Studies from our groups and others suggested that signal transduction in primary erythroid cells differs considerably from that in cell lines or primary cells from other species. In this review, we summarize results of these studies with emphasis on possible implications for hematotherapy.

Animals↗
PubMed2001

Improvement of the precision in CFU-GM and BFU-E counting by flow cytometry-based standardization of short-term culture assays.

The counting of colony-forming units granulocyte-macrophage (CFU-GM) and burst-forming units erythrocyte (BFU-E) provides substantial in vitro information about the graft quality after peripheral stem cell transplantation (PBSCT). By using different techniques for culturing and scoring, high inter- and intralaboratory coefficients of variation (CV) are frequently reported. We minimized the imprecision by using flow cytometry-based incorporation of constant numbers of CD34(+) cells per culture dish instead of the formerly used mononuclear cells. Our results show acceptable CVs for CFU-GM (12.3%) and for BFU-E (13.3%) based on this seeding technique, which contributes to fulfilling the demands of a quality assurance system in stem cell laboratories.

Antigens, CD34↗
PubMed2001

Developmental regulation of DNA replication timing at the human beta globin locus.

The human beta globin locus replicates late in most cell types, but becomes early replicating in erythroid cells. Using FISH to map DNA replication timing around the endogenous beta globin locus and by applying a genetic approach in transgenic mice, we have demonstrated that both the late and early replication states are controlled by regulatory elements within the locus control region. These results also show that the pattern of replication timing is set up by mechanisms that work independently of gene transcription.

Animals↗
PubMed2000

Hemopoiesis in healthy old people and centenarians: well-maintained responsiveness of CD34+ cells to hemopoietic growth factors and remodeling of cytokine network.

In vitro hemopoiesis and hemopoietic cytokines production were evaluated in 9 centenarians (median age 100.5 years, age range: 100-104 years), 10 old people (median age: 71 years, age range: 66-73 years), and 10 young people (median age: 35 years, age range: 30-45 years), all carefully selected for their healthy status. The main findings were the following: (i) a trend towards a decreased absolute number of CD34+ progenitor cells in the peripheral blood of old people and centenarians, in comparison to young subjects; (ii) a well-preserved capability of CD34+ cells from old people and centenarians to respond to hemopoietic cytokines, and to form erythroid (BFU-E), granulocyte-macrophagic (CFU-GM), and mixed colonies (CFU-GEMM) in a way (number, size, and morphology) indistinguishable from that of young subjects; (iii) an age-related decreased in vitro production of granulocyte-macrophagic colony-stimulating factor (GM-CSF) and a decreased production of interleukin-3 (IL-3) in centenarians by phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC); (iv) a linear increase of the serum level of stem cell factor (SCF), measured in the above-mentioned subjects and in 65 additional subjects, including 4 centenarians. These data suggest that basal hematopoietic potential is well preserved in healthy centenarians, and that the hemopoietic cytokine network undergoes a complex remodeling with age.

Adult↗
PubMed1994

In vivo-in vitro correlation of myelotoxicity of 9-methoxypyrazoloacridine (NSC-366140, PD115934) to myeloid and erythroid hematopoietic progenitors from human, murine, and canine marrow.

BACKGROUND: 9-Methoxypyrazoloacridine (PZA) is an anticancer agent that shows selectivity of action for carcinomas over leukemias. It also has nearly equal potency against cycling and quiescent or hypoxic and normoxic target cells. Phase I trials of PZA in humans are nearing completion. PURPOSE: This study was conducted to determine (a) if PZA is directly inhibitory to hematopoietic cells and, if it is, to characterize the inhibition pharmacodynamically, (b) whether species-specific differences in direct toxicity could explain differences in myelosuppression in mice, dogs, and humans, and (c) whether in vitro data correlate with in vivo myelosuppression data. METHODS: In vitro clonogenic assays of hematopoietic progenitors of myeloid and erythroid lineages from human, canine, and murine femoral marrow were used to measure the direct toxicity of PZA. Results from these assays were compared on an area-under-the-curve (AUC) basis to clinical myelosuppression data. RESULTS: On the basis of maximum tolerated concentrations, canine hematopoietic progenitors are most susceptible to PZA, followed by human and then murine progenitors. We found no difference in susceptibility to PZA toxicity between the human progenitors of myeloid and erythroid lineages. Both concentration and duration of exposure contribute to the in vitro toxicity of PZA. In contrast to antimetabolites, the in vitro toxicity of PZA could be minimized at a given AUC by lowering drug concentration and prolonging the period of exposure. On an AUC basis, the in vitro data are consistent with limited in vivo myelosuppression data from preclinical models and correlate with neutropenia data from a phase I trial. CONCLUSIONS: PZA directly inhibits hematopoietic progenitors, an action that is responsible for the myelosuppression observed in humans. Human marrow appears able to compensate for the loss of up to 35% of its myeloid progenitors, in that peripheral neutrophil counts remain unchanged at that level of loss. Although in vivo studies show that prolonged infusion reduces myelosuppression at a given total dose in both rodent and canine models, pharmacokinetic differences make it unlikely that this approach will benefit human patients. IMPLICATIONS: The in vitro data quantitatively predict the AUCs at maximum tolerated dose in preclinical models and human patients. Thus, in vitro clonogenic assays of myelotoxic agents can provide data that make both preclinical toxicology testing and clinical trial planning and interpretation more efficient and accurate.

Acridines↗
PubMed1992

Severe in vitro inhibition of erythropoiesis and transient stimulation of granulopoiesis after bone-marrow infection with eight different HIV-2 isolates.

OBJECTIVE: To correlate severe anaemia and frequent neutropenia in HIV-2-infected patients with an inhibitory effect on bone-marrow progenitors common to several HIV-2 isolates. DESIGN: The effects of eight HIV-2 isolates on early (BFU-E) and late (CFU-E) erythroid progenitors and on granulomonocytic (CFU-GM) progenitors, produced in long-term bone-marrow cultures (LTBMC), were studied. METHODS: Absolute numbers of BFU-E, CFU-E and CFU-GM per culture flask were calculated weekly for each HIV-2-infected LTBMC using semi-solid clonogenic assays, and compared with those obtained in mock-infected LTBMC. Levels of significance for comparisons were determined by an analysis of variance (ANOVA). RESULTS: Pooled data from 24 series of LTBMC (three series for each HIV-2 isolate) revealed 80 and 100% inhibition of BFU-E and CFU-E on days 6 and 12 of LTBMC, respectively, while transient stimulation of CFU-GM was observed between days 14 and 20 of LTBMC, followed by total inhibition on day 30. CONCLUSIONS: These results confirm a direct inhibitory effect of HIV-2 on in vitro haematopoiesis. The similar pattern of erythroid progenitor inhibition obtained from seven out of eight isolates suggests that the inhibitory effect on erythropoiesis is a feature common to a large number of HIV-2 isolates, which correlates with clinical findings.

Blotting, Southern↗
PubMed2002

New insights into erythropoiesis.

Commitment of hematopoietic cells to the erythroid lineage involves the actions of several transcription factors, including TAL1, LMO2, and GATA-2. The differentiation of committed erythroid progenitor cells involves other transcription factors, including NF-E2 and EKLF. Upon binding erythropoietin, the principal regulator of erythropoiesis, cell surface erythropoietin receptors dimerize and activate specific intracellular kinases, including Janus family tyrosine protein kinase 2, phosphoinositol-3 kinase, and mitogen-activated protein kinase. Important substrates of these kinases are tyrosines in the erythropoietin receptors themselves and the signal transducer and transcription activator proteins. Erythropoietin prevents erythroid cell apoptosis. Some of the apoptotic tendency of erythroid cells can be attributed to proapoptotic molecules produced by hematopoietic cells, macrophages, and stromal cells. Cell divisions accompanying terminal erythroid differentiation are finely controlled by cell cycle regulators, and disruption of these terminal divisions causes erythroid cell apoptosis. In reticulocyte maturation, regulated degradation of internal organelles involves a lipoxygenase, whereas survival requires the antiapoptotic protein Bcl-x.

Animals↗
PubMed2005

The impact of a hypercatecholamine state on erythropoiesis following severe injury and the role of IL-6.

BACKGROUND: Severe traumatic injury can lead to hemorrhagic shock-induced bone marrow (BM) dysfunction resulting in persistent anemia. The hypercatacholamine state that accompanies severe injury has been shown to impact the growth of erythroid progenitors. IL-6 has a role both in the acute phase response of trauma and has been implicated in the development of anemia. The aim of this study was to investigate the severity of a hyper-adrenergic stimulus on pluripotent progenitors (GEMM-CFU) as well as erythroid progenitors (BFU-E and CFU-E) and the potential regulatory role of IL-6. METHODS: Normal human BM mononuclear cells were isolated and erythropoiesis was assessed by the growth of GEMM-CFU, BFU-E and CFU-E in the presence of adrenergic agonists, norepinephrine (NE) and epinephrine (EPI), at increasing concentrations. Similarly, normal BM stroma cells were grown to confluence then incubated with NE and EPI. Supernatant was harvested and IL-6 levels were determined using ELISA. RESULTS: Under physiologic conditions (10(-7) M), NE and EPI increase BFU-E and CFU-E growth (374% and 177% versus 100% control). At severe stress levels (10(-3) M), NE and EPI completely inhibited BFU-E and CFU-E growth (5% and 4% versus 100% control). GEMM-CFU growth was increased by NE and not EPI at 10(-7) M. The presence of NE and EPI increased IL-6 levels in a dose-dependent fashion. CONCLUSIONS: The proliferative effect of adrenergic agonists at physiologic levels on normal erythropoiesis begins early during erythroid differentiation. At severe stress levels, BFU-E and CFU-E growth is inhibited. The erythropoietic dysfunction and resultant anemia seen following severe injury may be due to the presence of a severe hypercatecholamine state and may be mediated by IL-6.

Bone Marrow Cells↗
PubMed1991

Treatment of refractory pure red cell aplasia with cyclosporine A: disappearance of IgG inhibitor associated with clinical response.

Remissions were obtained in 6/9 evaluable patients with pure red cell asplasia (PRCA) refractory to other immunosuppressive agents who were treated with cyclosporine A (CsA). Four of these patients have remained in continuous remission off all treatment for 4-19 months. Another patient who stopped CsA abruptly relapsed, but responded to reinstitution of therapy. The sixth patient died of a cerebrovascular accident while in remission on a low dose of CsA. Acute side effects were minimal and were responsive to dose reduction. One patient developed a lymphoma while in an unmaintained remission, and one patient who did not respond to CsA was found to have a lymphoma approximately a year after stopping treatment. In vitro studies of autologous erythroid progenitors in a patient with an IgG inhibitor of erythropoiesis showed a reduction of autoantibody associated with the response to CsA. The antigen to which this inhibitor is directed was expressed only during the marrow erythroid burst-forming unit (BFU-E) period of erythroid differentiation. CsA can induce sustained remissions in cases of PRCA refractory to other multiple agents, and these remissions may be associated with a reduction in autoantibody to erythroid progenitor cells. Further studies of patients with PRCA who respond to CsA may lead to an improved understanding of this disorder.

Adolescent↗
PubMed1991

Lymphohaematopoietic chimaerism after bone marrow transplantation for chronic myeloid leukaemia: results of simultaneous cytogenetic analyses on T-cell colonies, myeloid, and erythroid progenitor cells.

Various lymphohaematopoietic compartments represented by cells from T-cell colonies, myeloid progenitor cells (CFU-GM), erythroid progenitor cells (BFU-E), and bone marrow after short-term culture (BM) have simultaneously been analysed in 15 patients receiving 17 bone marrow transplants for Philadelphia chromosome (Ph) positive chronic myeloid leukaemia (CML) or acute lymphoblastic leukaemia (ALL). The marrow grafts were not T-cell depleted. Ten patients without relapse did not show any myeloid cells of host origin until their last follow-up or until death. However, in four of these patients single lymphoid host cells not carrying the Ph chromosome were found after BMT without clinical consequences. In patients with cytogenetic or haematological relapse Ph positive metaphases were first detected in any of the progenitor cell compartments along with residual donor cells in two of three patients. BM became Ph positive after various time intervals. Another patient with CML became Ph positive in all compartments investigated at the same time. The only patient with Ph positive ALL remained completely Ph negative also when haematological and clinical relapse was evident. All patients with relapse exhibited complex clonal and non-clonal chromosomal aberrations at the time of recurrence of the Ph chromosome. Such abnormalities not identical to those usually found with evolution of the disease and preferentially occurring in progenitor cells preceded the reappearance of Ph positive metaphases in one of our patients.

Adolescent↗
PubMed1995

B lymphocytes with latent EBV infection appearing in long-term bone marrow cultures (HLTBMCs) from haematological patients induce lysis of stromal microenvironment.

Human long-term bone marrow cultures (HLTBMCs) are a valuable in vitro model for studying the role of the haemopoietic microenvironment. Here we report the spontaneous appearance of EBV-positive B cells in 6/40 HLTBMCs from patients with various haematological diseases after 3-5 months of culture. After subcultivation of these cells, a novel type of cell line could be characterized, which displayed surface markers and morphological features typical for EBV transformed B-cell lines. As the deproteinized and ultrafiltrated culture supernatants of these cell lines were found to contain an agent with stroma toxic properties, they were termed SSB lines (stroma-toxic-agent-secreting B-cell lines). This agent also exhibited a colony-inhibitory activity on in vitro myelopoiesis and erythropoiesis. These properties are typical for the two polyamines spermine and spermidine which were detected at elevated levels in the culture supernatants of SSB lines. The hypothesis that latent presence of EBV in bone marrow may induce an increased synthesis of spermine and spermidine, which are known to be associated with malignant haematological diseases and bone marrow aplasia, is discussed.

B-Lymphocytes↗
PubMed2005

The expression of minichromosome maintenance protein-2 in normal and abnormal megakaryocytes and comparison with the proliferative marker Ki-67.

The minichromosome maintenance (Mcm) and Cdc6 proteins are important regulators of eucaryotic DNA replication. In most normal tissues, a similar proportion of cells express Mcm-2 and Ki-67. The present study showed that in both normal and abnormal states, the proportion of megakaryocytes expressing Mcm-2 is roughly seven times as many as those that express Ki-67. This is likely to be related to the process of endomitosis and endoreduplication. We also demonstrated that a significantly lower proportion of megakaryocytes in myelodysplastic syndrome express Mcm-2. These findings provide new insights into megakaryocyte biology.

Bone Marrow↗
PubMed2004

In vivo effects of interleukin-17 on haematopoietic cells and cytokine release in normal mice.

In order to gain more insight into mechanisms operating on the haematopoietic activity of the T-cell-derived cytokine, interleukin-17 (IL-17) and target cells that first respond to its action in vivo, the influence of a single intravenous injection of recombinant mouse IL-17 on bone marrow progenitors, further morphologically recognizable cells and peripheral blood cells was assessed in normal mice up to 72 h after treatment. Simultaneously, the release of IL-6, IL-10, IGF-I, IFN-gamma and NO by bone marrow cells was determined. Results showed that, in bone marrow, IL-17 did not affect granulocyte-macrophage (CFU-GM) progenitors, but induced a persistant increase in the number of morphologically recognizable proliferative granulocytes (PG) up to 48 h after treatment. The number of immature erythroid (BFU-E) progenitors was increased at 48 h, while the number of mature erythroid (CFU-E) progenitors was decreased up to 48 h. In peripheral blood, white blood cells were increased 6 h after treatment, mainly because of the increase in the number of lymphocytes. IL-17 also increased IL-6 release and NO production 6 h after administration. Additional in vitro assessment on bone marrow highly enriched Lin- progenitor cells, demonstrated a slightly enhancing effect of IL-17 on CFU-GM and no influence on BFU-E, suggesting the importance of bone marrow accessory cells and secondary induced cytokines for IL-17 mediated effects on progenitor cells. Taken together, these results demonstrate that in vivo IL-17 affects both granulocytic and erythroid lineages, with more mature haematopoietic progenitors responding first to its action. The opposite effects exerted on PG and CFU-E found at the same time indicate that IL-17, as a component of a regulatory network, is able to intervene in mechanisms that shift haematopoiesis from the erythroid to the granulocytic lineage.

Animals↗
PubMed1995

Structural and signaling requirements for BCR-ABL-mediated transformation and inhibition of apoptosis.

BCR-ABL is a deregulated tyrosine kinase expressed in Philadelphia chromosome-positive human leukemias. Prolongation of hematopoietic cell survival by inhibition of apoptosis has been proposed to be an integral component of BCR-ABL-induced chronic myelogenous leukemia. BCR-ABL elicits transformation of both fibroblast and hematopoietic cells and blocks apoptosis following cytokine deprivation in various factor-dependent cells. To elucidate the mechanisms whereby BCR-ABL induces transformation and blocks apoptosis in hematopoietic cells, we examined the biological effects of expression of a series of BCR-ABL mutants. Single amino acid substitutions in the GRB2 binding site (Y177F), Src homology 2 domain (R552L), or an autophosphorylation site in the tyrosine kinase domain (Y793F) do not diminish the antiapoptotic and transforming properties of BCR-ABL in hematopoietic cells, although these mutations were previously shown to drastically reduce the transforming activity of BCR-ABL in fibroblasts. A BCR-ABL molecule containing all three mutations (Y177F/R552L/Y793F) exhibits a severe decrease in transforming and antiapoptotic activities compared with the wild-type BCR-ABL protein in 32D myeloid progenitor cells. Ras is activated, the SHC adapter protein is tyrosine phosphorylated and binds GRB2, and myc mRNA levels are increased following expression of all kinase active BCR-ABL proteins with the exception of the Y177F/R552L/Y793F BCR-ABL mutant in 32D cells. We propose that BCR-ABL uses multiple pathways to activate Ras in hematopoietic cells and that this activation is necessary for the transforming and antiapoptotic activities of BCR-ABL. However, Ras activation is not sufficient for BCR-ABL-mediated transformation. A BCR-ABL deletion mutant (delta 176-427) that activates Ras and blocks apoptosis but has severely impaired transforming ability in 32D cells has been identified. These data suggest that BCR-ABL requires additional signaling components to elicit tumorigenic growth which are distinct from those required to block apoptosis.

Adaptor Proteins, Signal Transducing↗
PubMed1998

AP1 regulation of proliferation and initiation of apoptosis in erythropoietin-dependent erythroid cells.

The transcription factor AP1 has been implicated in the induction of apoptosis in cells in response to stress factors and growth factor withdrawal. We report here that AP1 is necessary for the induction of apoptosis following hormone withdrawal in the erythropoietin (EPO)-dependent erythroid cell line HCD57. AP1 DNA binding activity increased upon withdrawal of HCD57 cells from EPO. A dominant negative AP1 mutant rendered these cells resistant to apoptosis induced by EPO withdrawal and blocked the downregulation of Bcl-XL. JunB is a major binding protein in the AP1 complex observed upon EPO withdrawal; JunB but not c-Jun was present in the AP1 complex 3 h after EPO withdrawal in HCD57 cells, with a concurrent increase in junB message and protein. Furthermore, analysis of AP1 DNA binding activity in an apoptosis-resistant subclone of HCD57 revealed a lack of induction in AP1 DNA binding activity and no change in junB mRNA levels upon EPO withdrawal. In addition, we determined that c-Jun and AP1 activities correlated with EPO-induced proliferation and/or protection from apoptosis. AP1 DNA binding activity increased over the first 3 h following EPO stimulation of HCD57 cells, and suppression of AP1 activity partially inhibited EPO-induced proliferation. c-Jun but not JunB was present in the AP1 complex 3 h after EPO addition. These results implicate AP1 in the regulation of proliferation and survival of erythroid cells and suggest that different AP1 factors may play distinct roles in both triggering apoptosis (JunB) and protecting erythroid cells from apoptosis (c-Jun).

Apoptosis↗
PubMed1982

The myeloproliferative sarcoma virus causes transformation or erythroid progenitor cells in vitro.

The myeloproliferative sarcoma virus induces spleen focus formation in vivo and transforms fibroblasts in vitro. We showed in this study that in vitro infection of spleen or bone marrow cells from susceptible mice with the myeloproliferative sarcoma virus leads to the formation of erythroid bursts. Under optimal conditions erythroid bursts formed in the absence of added erythropoietin, but the addition of as little as 0.05 U of erythropoietin per ml to infected cultures resulted in a significant increase in numbers of erythroid bursts and the proportion of hemoglobinized cells. A comparison of the kinetics of burst formation and the size of the induced bursts with those induced with Friend virus suggested that either sarcoma virus such as the myeloproliferative sarcoma virus or the target cells for the two viruses were not the same. Density characterization and heat lability studies indicated that the increased erythroid proliferation in vitro was a virus-induced event, but the possibility that the induced erythroid burst formation is mediated via interaction with a nonerythroid target cell and subsequent release of a soluble factor cannot be ruled out.

Animals↗
PubMed2003

Highly restricted localization of RNA polymerase II within a locus control region of a tissue-specific chromatin domain.

RNA polymerase II (Pol II) can associate with regulatory elements far from promoters. For the murine beta-globin locus, Pol II binds the beta-globin locus control region (LCR) far upstream of the beta-globin promoters, independent of recruitment to and activation of the betamajor promoter. We describe here an analysis of where Pol II resides within the LCR, how it is recruited to the LCR, and the functional consequences of recruitment. High-resolution analysis of the distribution of Pol II revealed that Pol II binding within the LCR is restricted to the hypersensitive sites. Blocking elongation eliminated the synthesis of genic and extragenic transcripts and eliminated Pol II from the betamajor open reading frame. However, the elongation blockade did not redistribute Pol II at the hypersensitive sites, suggesting that Pol II is recruited to these sites. The distribution of Pol II did not strictly correlate with the distributions of histone acetylation and methylation. As Pol II associates with histone-modifying enzymes, Pol II tracking might be critical for establishing and maintaining broad histone modification patterns. However, blocking elongation did not disrupt the histone modification pattern of the beta-globin locus, indicating that Pol II tracking is not required to maintain the pattern.

Acetylation↗