Tyrphostin AG957, a tyrosine kinase inhibitor with anti-BCR/ABL tyrosine kinase activity restores beta1 integrin-mediated adhesion and inhibitory signaling in chronic myelogenous leukemia hematopoietic progenitors

Abnormal beta1 integrin receptor function may contribute to the continuous proliferation and abnormal circulation of malignant hematopoietic progenitors in chronic myelogenous leukemia (CML). Previous studies suggest that abnormal integrin function in CML progenitors is related to the presence of the BCR/ABL oncogene. BCR/ABL may alter integrin function in CML by phosphorylating cytoskeletal and/or signaling proteins important for normal integrin function. We evaluated the effect of Tyrphostin AG957, a protein tyrosine kinase (PTK) inhibitor which has activity against the p210BCR/ABL kinase, on beta1 integrin function in CML progenitors. Incubation of CML marrow CD34+HLA-DR+ cells with Tyrphostin AG957 at concentrations that did not affect colony-forming cells (CFC) viability, but which partly inhibited p210BCR/ABL kinase activity, significantly increased CML CFC adhesion to stroma and alpha4beta1 and alpha5beta1 integrin binding fragments of fibronectin (FN). CML CFC proliferation, unlike that of normal CFC, is not inhibited following integrin receptor engagement with FN or anti-integrin antibodies. AG957 did not alter CML CFC proliferation by itself, but resulted in significant inhibition of CML CFC proliferation following integrin engagement. Another PTK inhibitor, Tyrphostin AG555, which does not have anti-p210BCR/ABL kinase activity, did not affect CML CFC adhesion or proliferation. Neither AG957 nor AG555 affected normal CFC adhesion or proliferation. In BCR/ABL expressing cells, AG957 partially inhibited phosphorylation of several proteins that are BCR/ABL PTK substrates and are involved in normal integrin signaling. These studies suggest that abnormal tyrosine phosphorylation may play an important role in defective integrin function in CML progenitors.     

Mobilization and homing of peripheral blood progenitors is related to reversible downregulation of alpha4 beta1 integrin expression and function

Despite the wide use of mobilized peripheral blood (PB) progenitor cells (PBPC) for clinical transplantation the mechanism(s) underlying their mobilization and subsequent engraftment are still unknown. We compared the adhesive phenotype of CD34(+) colony-forming cells (CFC) in bone marrow (BM) and PB of normal donors before and after administration of granulocyte colony-stimulating factor (G-CSF) for 5 d. G-CSF-mobilized PB CFC cells adhered significantly less to BM stroma, fibronectin, and to the alpha4 beta1 binding fibronectin peptide, CS1, because of decreased expression of the alpha4 integrin. Since incubation of BM CD34(+) cells for 4 d with G-CSF at concentrations found in serum of G-CSF- treated individuals did not affect alpha4-dependent adhesion, G-CSF may not be directly responsible for the decreased alpha4-mediated adhesion of PB CFC. Culture of G-CSF-mobilized PB CD34(+) cells with cytokines at concentrations found in BM stromal cultures upregulated alpha4 expression and restored adhesion of mobilized PB CFC to stroma, fibronectin, and CS1. Adhesion of cultured, mobilized PB CFC to stroma and CS1 could not be further upregulated by the beta1 activating antibody, 8A2. This indicates acquisition of a maximally activated alpha4 beta1 integrin once PB CFC have been removed from the in vivo mobilizing milieu. Thus, decreased alpha4 expression on CD34(+) CFC in PB may be responsible for the aberrant circulation of mobilized PB CD34(+) cells. Reexpression of a maximally activated alpha4 beta1 integrin on mobilized PB CFC removed from the mobilizing in vivo milieu may contribute to the early engraftment of mobilized PBPC.     

BCR/ABL- CD34(+)HLA-DR- progenitor cells in early chronic phase, but not in more advanced phases, of chronic myelogenous leukemia are polyclonal

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia (Ph) translocation and BCR/ABL gene rearrangement which occur in a pluripotent hematopoietic progenitor cell. Ph-negative (Ph-) hematopoiesis can be restored in vivo after treatment with -interferon or intensive chemotherapy, suggesting that normal stem and progenitor cells coexist with the Ph+ clone. We have previously shown that Ph- progenitors are highly enriched in the CD34(+)HLA-DR- fraction from early chronic phase (ECP) CML patients. Previous studies have suggested that the Ph-translocation represents a secondary clonal hit occurring in an already clonally mutated Ph- progenitor or stem cells, leaving the unanswered question whether Ph- CD34(+)HLA-DR- progenitors are normal. To show the clonal nature of Ph- CD34(+)HLA-DR- CML progenitors, we have compared the expression of BCR/ABL mRNA with X-chromosome inactivation patterns (HUMARA) in mononuclear cells and in CD34(+)HLA-DR+ and CD34(+)HLA-DR- progenitors in marrow and blood obtained from 11 female CML patients (8 in chronic phase and 3 in accelerated phase [AP] disease). Steady-state marrow-derived BCR/ABL mRNA-, CD34(+)HLA-DR- progenitors had polyclonal X-chromosome inactivation patterns in 2 of 2 patients. The same polyclonal pattern was found in the progeny of CD34(+)HLA-DR- derived long-term culture-initiating cells. Mobilization with intensive chemotherapy induced a Ph-, BCR/ABL mRNA- and polyclonal state in the CD34(+)HLA-DR- and CD34(+)HLA-DR+ progenitors from 2 ECP patients. In a third ECP patient, polyclonal CD34(+) cells could only be found in the first peripheral blood collection. In contrast to ECP CML, steady-state marrow progenitors in late chronic phase and AP disease were mostly Ph+, BCR/ABL mRNA+, and clonal. Further, in the majority of these patients, a Ph-, polyclonal state could not be restored despite mobilization with intensive chemotherapy. We conclude from these studies that CD34(+)HLA-DR- cells that are Ph- and BCR/ABL mRNA- are polyclonal and therefore benign. This population is suitable for autografting in CML.     

Chronic myelogenous leukemia: molecular and cellular aspects

Chronic myelogenous leukemia (CML) originates in a pluripotent hematopoietic stem cell of the bone marrow and is characterized by greatly increased numbers of granulocytes in the blood. Myeloid and other hematopoietic cell lineages are involved in the process of clonal proliferation and differentiation. After a period of 4-6 years the disease progresses to acute-stage leukemia. On the cellular level, CML is associated with a specific chromosome abnormality, the t(9; 22) reciprocal translocation that forms the Philadelphia (Ph) chromosome. The Ph chromosome is the result of a molecular rearrangement between the c-ABL proto-oncogene on chromosome 9 and the BCR (breakpoint cluster region) gene on chromosome 22. Most of ABL is linked with a truncated BCR. The BCR/ABL fusion gene codes for an 8-kb mRNA and a novel 210-kDa protein which has higher and aberrant tyrosine kinase activity than the normal c-ABL-coded counterpart. Phosphorylation of a number of substrates such as GAP, GRB-2, SHC, FES, CRKL, and paxillin is considered a decisive step in transformation. An etiological connection between BCR/ABL and leukemia is indicated by the observation that transgenic mice bearing a BCR/ABL DNA construct develop leukemia of B, T, and myeloid cell origin. CML cells proliferate and expand in an almost unlimited manner. Adhesion defects in bone marrow stromal cells have been proposed to explain the increased number of leukemic cells in the peripheral blood. However, findings of our laboratory have shown that the BCR/ABL chimeric protein that is expressed in transfected cells may, under certain conditions, also increase the adhesion to fibronectin via enhanced expression of integrin. Our previous immunocytological studies on the expression of beta1 and beta2 integrins have found no qualitative differences between normal and CML hematopoietic cells in vitro. Even long-term-cultured CML bone marrow or blood cells continuously express those adhesion molecules that are characteristic of the cytological type. Recent experiments indicate that certain early CML progenitors may adhere to the stromal layer in vitro similarly to their normal counterparts. They cannot be completely removed by long-term culture on allogeneic stromal cells. At present, the only curative therapy is transplantation of allogeneic hematopoietic stem cells. Based on the molecular and cellular state of knowledge of CML, new therapies are being developed. BCR/ABL antisense oligonucleotides, inhibitors of tyrosine kinase, peptide-specific adoptive immunotherapy or peptide vaccination, and restoration of hematopoiesis by autologous stem cell transplantation following CML cell purging are examples of important approaches to improving CML treatment.     

Reemergence of gentamicin-susceptible strains of methicillin-resistant Staphylococcus aureus: roles of an infection control program and changes in aminoglycoside use

We have tested the effect of stromal cells on the proliferation in long- and short-term cultures of primitive (Thy-1+, CD34+, CD33-, CD38- , HLA-DR , adherent in vitro and quiescent in vivo) progenitors in normal human bone marrow. These primitive cells produce granulocyte-macrophage colony-forming cells (CFU-GM) that are measured in secondary clonogenic assays. Addition of stromal cells to normal adherent haemopoietic progenitor cells reduced CFU-GM production by 80% (P =0.0002) after 1 week of incubation. In long-term culture (LTC), in the presence of stroma. the normal adherent cells did not produce significant numbers of CFU-GM until 3-4 weeks later which suggests that stromal cells reduce the probability of quiescent cell activation. This effect could not be attributed to soluble inhibitory factors and was specific to stroma grown with, rather than without, methylprednisolone. It was blocked by heparanase (H'ase) II treatment of stromal cells, by phosphatidylinositol-specific phospholipase C (PI-PLC) treatment of progenitor cells, by antibody blocking of beta1 integrin molecules or by exposure to glucose/N-acetyl-D-glucosamine/alpha-methyl-D-mannoside, but not by exposure to galactose or fructose. Moreover, these interventions enabled the progenitor cells to respond to stimulatory factors in the culture supernatant. We interpret these results as support for a model involving primitive progenitor cell binding to stroma by PI-CAM/HS, beta1 integrin activation via lectin-like interactions and the transduction of signals which reduce the ability of primitive cells to respond to ambient stimulators. This model provides a mechanism for the maintenance of the quiescent state of stem cells by adhesion to stromal cells.     

Identification of allergens in oilseed rape (Brassica napus) pollen

Monoclonal antibodies to very late antigen 4 (VLA-4) recognize the alpha4beta1 integrin receptor. This monoclonal antibody blocks the adhesion between early hematopoietic progenitor cells (CD34-selected cells) and stromal cells when added to cultures of these cells. Addition of the VLA-4 monoclonal antibody to cultures of stromal cells and CD34-selected cells was shown to induce apoptosis of CD34-selected cells in these CD34-selected cell/stromal cell cocultures, as measured by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling method. In contrast to these experiments with early hematopoietic progenitor cells (CD34+), the level of adhesion between more differentiated cells (unfractionated hematopoietic cells) and stromal cells was not significantly altered by addition of the anti-VLA-4 monoclonal antibody. Similarly, the level of apoptosis of unfractionated hematopoietic cells was not significantly increased by the addition of anti-VLA-4 monoclonal antibody to cultures of the latter cells with stromal cells. The binding of the unfractionated cells is less than that of the CD34-selected cells. Given that there is no difference between the alpha4beta1 integrin expression level of the early and late myeloid cells, there may be a difference in the functional state of the integrin between the early and late myeloid cells. We also show that CD34+-selected precursor cells proliferate at a higher rate when these cells are plated on recombinant vascular cell adhesion molecule 1 molecules. These data indicate that the alpha4beta1 integrin receptor (VLA-4) plays a central role in the apoptosis rescue function that results from the anchorage-dependent growth of the CD34-selected early hematopoietic cells on stromal cells. The data suggest that these apoptosis rescue pathways have less significance as the cells mature and become anchorage independent in their growth. These data should assist in the design of systems for the ex vivo proliferation and transduction of early hematopoietic cells for genetic therapy.     

MCP-1, not MIP-1alpha, is the endogenous chemokine that cooperates with TGF-beta to inhibit the cycling of primitive normal but not leukemic (CML) progenitors in long-term human marrow cultures

The long-term culture (LTC) system has been useful for analyzing mechanisms by which stromal cells regulate the proliferative activity of primitive normal, but not chronic myeloid leukemia (CML), hematopoietic progenitor cells. In previous studies, we identified two endogenous inhibitors in this system. One is transforming growth factor-beta (TGF-beta), which is equally active on primitive normal and CML progenitors. The other we now show to be monocyte chemoattractant protein-1 (MCP-1). Thus, MCP-1, when added to LTC, blocked the activation of primitive normal progenitors but did not arrest the cycling of primitive CML progenitors. Moreover, the endogenous inhibitory activity of LTC stromal layers could be overcome by the addition of neutralizing antibodies to MCP-1, but not to macrophage inflammatory protein-1alpha (MIP-1alpha). However, neither of these antibodies antagonized the inhibitory activity of NAc-Ser-Asp-Lys-Pro (AcSDKP) on primitive normal but not CML progenitor cycling in this system. Moreover, none of six other -C-C- or -C-X-C- chemokines, previously shown to inhibit primitive normal human CFC proliferation in semisolid assays, were found to act as negative regulators when added to normal LTC. These results provide further support for the concept that primitive CML progenitor cell proliferation is deregulated when these cells are exposed to limiting concentrations of multiple inhibitors, only some of which have differential actions on normal and Ph+/BCR-ABL+ cells.     

Thrombopoietin and erythropoietin activate inside-out signaling of integrin and enhance adhesion to immobilized fibronectin in human growth-factor-dependent hematopoietic cells

Erythropoietin (EPO) and thrombopoietin (c-MPL ligand; TPO) are structurally similar cytokines and support respectively, the proliferation and differentiation for erythroid and megakaryocytic lineages, as well as more primitive progenitors. We studied the effect of these cytokines on the induction of adhesion of human growth-factor-dependent hematopoietic cells to immobilized fibronectin, which is a main component of the extracellular matrix in the bone marrow. MO7ER cells that are genetically engineered to express human EPO receptor and MO7e cells that express endogenous c-MPL were used. Stimulation with either TPO or EPO induced rapid increases in adhesion of M07ER cells to fibronectin without apparent change of expression of integrins. Experiments with inhibitory monoclonal antibodies (mAbs) demonstrated that CD41, which has been reported to be involved in TPO-induced adhesion of megakaryocytic cells, is not responsible for this enhanced adhesion. Anti-beta 1 integrin mAb inhibited adhesion completely, while inhibition by anti-alpha 4 integrin mAb and anti-alpha 5 integrin mAb was partial. Combination of anti-alpha 4 mAb plus anti-alpha 5 mAb completely abolished adhesion, as did anti-beta 1 mAb, suggesting that the adhesion is mediated by both alpha 4 beta 1 and alpha 5 beta 1 integrins. Experiments using inhibitors suggested that ligand binding followed by activation of intracellular tyrosine kinases along with PI3-kinase activation is required. After stimulation of M07ER cells with either TPO or EPO, fibronectin-attached cells, but not cells in suspension, showed tyrosine phosphorylation of focal adhesion kinase, which plays a central role in integrin-mediated signaling. These data suggest that TPO and EPO might be involved in homing/migration to the bone marrow microenvironment by hematopoietic cells that express corresponding receptors.     

Proliferation and survival of mammary carcinoma cells are influenced by culture conditions used for ex vivo expansion of CD34(+) blood progenitor cells

Malignant cell contamination in autologous transplants is a potential origin of tumor relapse. Ex vivo expansion of CD34(+) blood progenitor cells (BPC) has been proposed as a tool to eliminate tumor cells from autografts. To characterize the influence of culture conditions on survival, growth, and clonogenicity of malignant cells, we isolated primary mammary carcinoma cells from pleural effusions and ascites of patients with metastatic breast cancer and cultured them in the presence of stem cell factor (SCF), interleukin-1beta (IL-1beta), IL-3, IL-6, and erythropoietin (EPO), ie, conditions previously shown to allow efficient ex vivo expansion of CD34(+) BPC. In the presence of serum, tumor cells proliferated during a 7-day culture period and no significant growth-modulatory effect was attributable to the presence of hematopoietic growth factors. When transforming growth factor-beta1 (TGF-beta1) was added to these cultures, proliferation of breast cancer cells was reduced. Expansion of clonogenic tumor cells was seen in the presence of SCF + IL-1beta + IL-3 + IL-6 + EPO, but was suppressed by TGF-beta1. Cocultures of tumor cells in direct cellular contact with hematopoietic cells showed that tumor cell growth could be stimulated by ex vivo expanded hematopoietic cells at high cell densities (5 x 10(5)/mL). In contrast, culture under serum-free conditions resulted in death of greater than 90% of breast cancer cells within 7 days and a further decrease in tumor cell numbers thereafter. In the serum-free cultures, hematopoietic cytokines and cellular contact with CD34(+) BPC could not protect the tumor cells from death. Therefore, ex vivo expansion of CD34(+) BPC in serum-free medium provides an environment for efficient purging of contaminating mammary carcinoma cells. These results have clinical significance for future protocols in autologous progenitor cell transplantation in cancer patients.     

Human CD34(+) bone marrow cells regulate stromal production of interleukin-6 and granulocyte colony-stimulating factor and increase the colony-stimulating activity of stroma

Cytokines produced by stromal cells induce the proliferation and differentiation of hematopoietic cells in the marrow microenvironment. We hypothesized that cross-talk between hematopoietic cells at different stages of differentiation and stromal cells influences stromal cytokine production and is responsible for maintaining steady-state hematopoiesis and responding to stress situations. We show that coculture of primitive CD34(+) cells in contact with or separated by a transwell membrane from irradiated human bone marrow stromal layers induces a fourfold to fivefold increase in interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) levels in the stromal supernatant (SN) during the first week. Levels of both cytokines decreased to baseline after coculture of CD34(+) cells for 3 to 5 weeks. Coculture of more mature CD15(+)/CD14(-) myeloid precursors induced only a transient 1.5- to 2-fold increase in IL-6 and G-CSF at 48 hours. Neither CD34(+) nor CD15(+)/CD14(-) cells produced IL-6, G-CSF, IL-1beta, or tumor necrosis factor alpha. When CD34(+) cells were cultured in methylcellulose medium supplemented with cytokines at concentrations found in stromal SN or supplemented with stromal SN, a fourfold to fivefold increase in colony formation was seen over cultures supplemented with erythropoietin (EPO) only. When cultures were supplemented with the increased concentrations of IL-6 and G-CSF detected in cocultures of stroma and CD34(+) cells or when CD34(+) cells were cocultured in methylcellulose medium in a transwell above a stromal layer, a further increase in the number and size of colonies was seen. The colony-forming unit-granulocyte-macrophage-stimulating activity of stromal SN was neutralized by antibodies against G-CSF or IL-6. These studies indicate that primitive CD34(+) progenitors provide a soluble positive feedback signal to induce cytokine production by stromal cells and that the observed increase in cytokine levels is biologically relevant.     

The sialomucin CD164 (MGC-24v) is an adhesive glycoprotein expressed by human hematopoietic progenitors and bone marrow stromal cells that serves as a potent negative regulator of hematopoiesis

Mucin-like molecules represent an emerging family of cell surface glycoproteins expressed by cells of the hematopoietic system. We report the isolation of a cDNA clone that encodes a novel transmembrane isoform of the mucin-like glycoprotein MGC-24, expressed by both hematopoietic progenitor cells and elements of the bone marrow (BM) stroma. This molecule was clustered as CD164 at the recent workshop on human leukocyte differentiation antigens. CD164 was identified using a retroviral expression cloning strategy and two novel monoclonal antibody (MoAb) reagents, 103B2/9E10 and 105.A5. Both antibodies detected CD164/MGC-24v protein expression by BM stroma and subpopulations of the CD34(+) cells, which include the majority of clonogenic myeloid (colony-forming unit-granulocyte-macrophage [CFU-GM]) and erythroid (blast-forming unit-erythroid [BFU-E]) progenitors and the hierarchically more primitive precursors (pre-CFU). Biochemical and functional characterization of CD164 showed that this protein represents a homodimeric molecule of approximately 160 kD. Functional studies demonstrate a role for CD164 in the adhesion of hematopoietic progenitor cells to BM stromal cells in vitro. Moreover, antibody ligation of CD164 on primitive hematopoietic progenitor cells characterized by the cell surface phenotype CD34(BRIGHT)CD38(-) results in the decreased recruitment of these cells into cell cycle, suggesting that CD164 represents a potent signaling molecule with the capacity to suppress hematopoietic cell proliferation.     

Inhibition of in vitro proliferation of chronic myelogenous leukemia progenitor cells by c-myb antisense oligodeoxynucleotides

Normal hematopoietic progenitors and acute myelogenous leukemia cells show a differential requirement for the encoded product of c-myb proto-oncogene for proliferation. To determine whether c-myb is also differentially required for the proliferation of hematopoietic progenitors of chronic myelogenous leukemia (CML), mononuclear cells derived from both chronic phase and blast crisis were exposed to c-myb antisense oligodeoxynucleotides and assayed for colony-forming ability. Exposure of CML-BC cells from 12 patients to c-myb antisense oligodeoxynucleotides resulted in significant (p<001) inhibition of leukemia colony formation (average inhibition 63%) and was accompanied by down-regulation of c-myb expression. Colonies derived from CML chronic phase progenitors were virtually unaffected in 10 cases, but down-regulation of c-myb expression was not detected. However, in studies conducted with CD34+ leukemia cells, a subset highly enriched for hematopoietic progenitors, colony formation was inhibited at both disease stages, whereas CFU-GM colony formation derived from normal CD34+ cells was not affected by exposure to c-myb antisense oligodeoxynucleotides. These data suggest that CML chronic phase and blast crisis progenitors are both sensitive to the inhibitory effects of c-myb antisense oligomers, and that the lack of inhibition in partially purified CML-chronic phase progenitors is probably due to inefficient penetration of oligodeoxynucleotides into the clonogenic cells. The preferential effect of c-myb antisense oligodeoxynucleotides on colonies arising from the compartment that includes CML-CD34+ progenitors likely reflects the expansion of a cell population with high proliferative potential and elevated c-myb mRNA levels.     

Effect of notoginsenoside R1 on the synthesis of components of the fibrinolytic system in cultured smooth muscle cells of human pulmonary artery

Manipulation of autologous bone marrow cells (BM) for transplantation in chronic myeloid leukemia (CML) to enrich for normal cells is a novel approach that may improve survival for patients not suitable for allogeneic transplantation. Limitations of this technique include the reported low frequency of normal stem cells in CML and the difficulties in obtaining sufficient BM for manipulation. To address these problems we compared the apheresis product with the diagnostic bone marrow at diagnosis as a source of primitive BCR/ABL-negative progenitors. We analyzed the CD34+ HLA-DR- and CD34+CD38(-) populations in five CML patients to evaluate the frequency of BCR-ABL-negative progenitors and pre-progenitors in these populations. Progenitor analysis was performed by RT-PCR of individual hemopoietic colonies from a standard CFU-GM assay. Analysis of pre-progenitors involved RT-PCR of secondary colonies derived from a stroma-free pre-CFU assay. Our results show variable levels of BCR-ABL-negative progenitors in the 34+DR- population but very low levels of BCR-ABL-negative progenitors in the 34+38- population in blood. Analysis of pre-progenitors from the 34+DR- fraction of peripheral blood (PB) and BM showed 80-100% and 85-100% of colonies were BCR-ABL negative at days 14 and 28, respectively. Analysis of pre-progenitors from the 34+38- fraction of PB and BM showed 23-100% and 42-100% of colonies were BCR-ABL negative at days 14 and 28, respectively. In summary, pre-progenitors from the 34+DR- and 34+38- populations are predominantly BCR-ABL negative in both marrow and blood at diagnosis. Apheresis product collected at diagnosis is a more abundant sources of BCR-ABL-negative pre-progenitors than BM. Thus, apheresis product could potentially be utilized as a source of BCR-ABL-negative stem cells in CML.     

Involvement of Fas-mediated apoptosis in the inhibitory effects of interferon-alpha in chronic myelogenous leukemia

Interferon-alpha (IFN-alpha) is an established treatment for chronic myelogenous leukemia (CML) in chronic phase, but the mechanism of its antileukemic activity is not clear. One possible mechanism of action might include the induction of apoptosis, and especially Fas-mediated cell killing may play an important role in the elimination of malignant cells. We investigated Fas receptor (Fas-R) expression and the consequences of Fas-R triggering in CML patients. Using two-color flow cytometry, we found a significantly higher number of Fas-R-expressing CD34+ cells in the bone marrow (BM) of CML patients compared with normal subjects. We have previously shown that IFN-gamma induces Fas-R expression on CD34+ cells; in this study, we investigated whether IFN-alpha induces Fas-R expression on CML progenitor cells. Dose-dependent induction of Fas-R expression was observed after IFN-alpha stimulation of CD34+ cells from CML BM. In methylcellulose culture, IFN-alpha alone at a therapeutic concentration showed only marginal antiproliferative effects on both normal and CML BM progenitors. In contrast, a Fas-R agonist, the anti-CD95 monoclonal antibody CH11, inhibited colony formation from normal progenitors, and the inhibition was even stronger on CML progenitors. When CML BM cells were cultured in the presence of IFN-alpha, Fas-R-mediated inhibition of colony growth was potentiated in a dose-dependent fashion, consistent with IFN-alpha induction of Fas-R expression. This functional effect did not require the presence of accessory cells, since similar results were obtained with purified CD34+ cells. In suspension cultures, we demonstrated that suppression of CML hematopoiesis by IFN-alpha and Fas-R agonist was exerted through Fas-R-mediated induction of apoptosis. Our findings suggest that the Fas-R/Fas-ligand system might be involved in the immunologic regulation of CML progenitor growth and that its effect can be amplified by IFN-alpha.     

New insights into the negative regulation of hematopoiesis by chemokine platelet factor 4 and related peptides

Platelet factor 4 (PF4) has been recognized as an inhibitor of myeloid progenitors. However, the mechanism of action of this chemokine remains poorly understood. The present study was designed to determine its structure/function relationship. A series of peptides overlapping the C-terminal and central regions of PF4 were analyzed in vitro for their action on murine hematopoietic progenitor growth to assess the minimal sequence length required for activity. The peptides p17-58 and p34-58 possessed an increased hematopoietic inhibitory activity when compared with PF4, whereas the shorter peptides p47-58 and p47-70 were equivalent to the native molecule and the peptide p58-70 was inactive. The PF4 functional motif DLQ located in 54-56 was required for the activity of these peptides. The peptide p34-58 impaired to a similar extent the growth of colony-forming unit-megakaryocyte (CFU-MK) as well as burst-forming unit-erythroid (BFU-E) and colony-forming unit-granulocyte-macrophage (CFU-GM), whereas PF4 was more active on CFU-MK. In the experiments using purified murine CD34(+) marrow cells, statistically significant inhibition induced by p34-58 was shown at concentrations of 2.2 nmol/L or greater for progenitors of the three lineages, whereas that induced by PF4 was seen at 130 nmol/L for CFU-MK and 650 nmol/L for CFU-GM and BFU-E, indicating that the p34-58 acts directly on hematopoietic progenitors and its activity is approximately 60- to 300-fold higher than PF4. The p34-58, unlike PF4, lacked affinity for heparin and its inhibitory activity could not be abrogated by the addition of heparin. In addition, an antibody recognizing p34-58 neutralized the activity of p34-58 but not whole PF4 molecule. These results demonstrate that PF4 contains a functional domain in its central region, which is independent of the heparin binding properties, and provide evidence for a model of heparin-dependent and independent pathways of PF4 in inhibiting hematopoiesis.     

HCA, an immunoglobulin-like adhesion molecule present on the earliest human hematopoietic precursor cells, is also expressed by stromal cells in blood-forming tissues

We have previously shown that the HCA/ALCAM (CD166) glycoprotein, a member of the immunoglobulin family that mediates both homophilic and heterophilic cell-cell adhesion, via the CD6 ligand, is expressed at the surface of all of the most primitive CD38(-/lo), Thy-1(+), rho123(lo), CD34(+) hematopoietic cells in human fetal liver and fetal and adult bone marrow. In the present report we show that HCA is also expressed by subsets of stromal cells in the primary hematopoietic sites that sequentially develop in the human embryo and fetus, ie, the paraaortic mesoderm, liver, thymus, and bone marrow. Adult bone marrow stromal cells established in vitro, including those derived from Stro-1(+) progenitors and cells from immortalized cell lines, express HCA. In contrast, no HCA expression could be detected in peripheral lymphoid tissues, fetal spleen, and lymph nodes. HCA membrane molecules purified from marrow stromal cells interact with intact marrow stromal cells, CD34(+) CD38(-) hematopoietic precursors, and CD3(+) CD6(+) peripheral blood lymphocytes. Finally, low but significant levels of CD6 are here for the first time detected at the surface of CD34(+) rho123(med/lo) progenitors in the bone marrow and in mobilized blood from healthy individuals. Altogether, these results indicate that the HCA/ALCAM surface molecule is involved in homophilic or heterophilic (with CD6) adhesive interactions between early hematopoietic progenitors and associated stromal cells in primary blood-forming organs.     

Thymosin beta4, inhibitor for normal hematopoietic progenitor cells

Thymosin beta4 (Tbeta4), isolated from the calf thymus fraction 5, has a ubiquitous localization and plays a pleiotropic role in both the immune and nonimmune systems. Because it contains at its N-terminal end the sequence of a known inhibitor of hematopoiesis, the acetylated tetrapeptide Ac-N-Ser-Asp-Lys-Pro (AcSDKP, Goralatide), we have assayed Tbeta4 on human hematopoietic cells. We demonstrate that it inhibits normal bone marrow progenitor cell growth; indeed, it decreased the growth of both granulo-macrophagic and erythroid progenitors and reduces their percentage in S phase. Furthermore, we show that Tbeta4 reduces both the clonogenicity and the cell proliferation of purified CD34+ cells induced by a combination of seven growth factors. Although Tbeta4's inhibitory effect is very similar to that of AcSDKP, we demonstrate, using neutralizing antibodies and a truncated form of Tbeta4 devoid of the AcSDKP sequence, that the inhibitory effect of Tbeta4 is not mediated by the sequence AcSDKP. These data indicate that Tbeta4 is a novel inhibitor for human normal hematopoietic progenitors.