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M W Long

Publications and source records attributed to M W Long.

At least 37 records · Page 2Linked to original sources
PubMed1995

Marrow-derived heparan sulfate proteoglycan mediates the adhesion of hematopoietic progenitor cells to cytokines.

Heparan sulfate proteoglycan (HS-PG), an important component of the human bone marrow extracellular matrix (ECM), is believed to influence hematopoietic progenitor cell development by binding and localizing growth factors to specific niches within the hematopoietic microenvironment. We utilized a model ECM system, which uses immobilized ECM proteins and/or cytokines and bone marrow populations enriched for human hematopoietic stem cell (HSC), to assess the effects of HS-PG on the development of primitive hematopoietic progenitor cells. HS-PG alone failed to bind hematopoietic progenitor cells cloned from bone marrow CD34+CD15-HLA-DR- cells, which are enriched for HSC. HS-PG alone failed to function as a mitogen. In sharp contrast, the interaction of HS-PG with either growth factors (interleukin-3 [IL-3] or stem cell factor/Kit ligand [KL] or an ECM protein (thrombospondin [TSP]) markedly influenced progenitor cell adherence. The binding of either IL-3 or KL to HS-PG resulted in a two-fold increase in attachment of the colony-forming unit-granulocyte/macrophage (CFU-GM), a 1.5-fold increase in attachment of the burst-forming unit-erythroid (BFU-E) and the high-proliferative-potential colony-forming cell (HPP-CFC), and a two- to three-fold increase in attachment of the colony-forming unit-granulocyte/erythroid/macrophage/megakaryocyte (CFU-GEMM) compared to localized growth factor alone. Attachment of the BFU-megakaryocyte (BFU-MK), however, was slightly reduced by the interaction of either IL-3 or KL with HS-PG. The interaction of HS-PG with TSP resulted in a two-fold increase in CFU-GM and CFU-GEMM attachment, while the attachment of BFU-E, HPP-CFC, and BFU-MK was unaltered. We conclude that HS-PG cooperatively interacts with both growth factors and ECM proteins to augment progenitor cell localization within the hematopoietic microenvironment.

Bone Marrow↗
PubMed1995

Cyclins and cell division kinases in megakaryocytic endomitosis.

Little is known concerning the mechanism by which megakaryocytes achieve their high levels of DNA content. Mature megakaryocytes show multiple 2-fold increases in DNA content, but the various levels of polyploidization exist within each of the morphologically recognizable classes. A number of studies have documented that the stimulatory actions of partially purified thrombopoietin or other cytokines on megakaryocyte DNA content both in vivo and in vitro. It is thus hypothesized that polyploidization is a crucial first step in megakaryocyte differentiation that is necessary for eventual cytoplasmic maturation and platelet production. Biochemically, there are 2 cell cycle regulatory points (either permissive or restrictive) which lead to polyploid DNA content in megakaryocytes; one regulatory point controls the increased DNA synthesis (presumably at the G1/S cell cycle boundary) and the other controls mitotic events, resulting in a single nucleus and an acytokinetic cell (the control point for this latter switch would be in early M-phase). Alterations in the biochemical control of these check points in other systems suggests that alterations in mitosis are among the first steps in endomitosis.

Animals↗
PubMed1993

Population heterogeneity among cells of the megakaryocyte lineage.

Understanding the developmental steps in megakaryocyte differentiation requires information regarding the microenvironmental influences which direct or permit the growth and differentiation of these cells. The megakaryocyte microenvironment, like other lineages, is a complex structure comprised of the various megakaryocytic cells, the extracellular matrix (ECM) surrounding them, and the hematopoietic stromal cells which elaborate both growth factors and ECM. As a result, definition of the minimal essential requirements for megakaryocyte development is difficult. The intricacies of megakaryocyte development are further complicated by the cellular heterogeneity of both mature megakaryocytes and their precursors, as well as a differential responsiveness of these cells to hematopoietic growth factors. This review focuses on defining the various subpopulations of megakaryocytic cells and examining their functional distinctions and in vitro responsiveness to various stimuli.

Cell Differentiation↗
PubMed1992

Human hematopoietic stem cell adherence to cytokines and matrix molecules.

The hematopoietic microenvironment is a complex structure in which stem cells, progenitor cells, stromal cells, growth factors, and extracellular matrix (ECM) molecules each interact to direct the coordinate regulation of blood cell development. While much is known concerning the individual components of this microenvironment, little is understood of the interactions among these various components or, in particular, the nature of those interactions responsible for the regional localization of specific developmental signals. We hypothesized that cytokines act together with ECM molecules to anchor stem cells within the microenvironment, thus modulating their function. In order to analyze matrix-cytokine-stem cell interactions, we developed an ECM model system in which purified stem cell populations and plastic-immobilized individual proteins are used to assess the role of various matrix molecules and/or cytokines in human hematopoietic cell development. Analysis of these interactions revealed that a single ECM protein, thrombospondin, in conjunction with a single cytokine (e.g., c-kit ligand), constitutes a developmental signal that synergistically modulates hematopoietic stem cell function.

Antigens, CD↗
PubMed1990

Developmental regulation of granulocytic cell binding to hemonectin.

Hemonectin (HN), a component of the bone marrow (BM) extracellular matrix which promotes adhesion of cells in the granulocytic lineage, was purified to near homogeneity and tested for its ability to mediate attachment of normal and leukemic cells of granulocytic lineage. Purified HN immobilized on plastic substrates promoted serum-free attachment of normal granulocyte/macrophage progenitor cells (CFC-GM), using an in situ attachment assay in which cell attachment is inhibited by specific polyclonal antisera. When unfractionated BM cells were allowed to attach to purified HN and stained in situ, HN preferentially bound cells at earlier stages of granulocytic differentiation. These observations were confirmed using cells of the HL-60 progranulocytic cell line which mirrored this differentiation-stage specific binding to HN. HN promoted attachment of 60% of uninduced HL-60 cells which were arrested at the progranulocyte stage, whereas only 15% of uninduced HL-60 cells attached to uncoated plastic and 4% to attached plastic coated with equal microgram quantities of bovine serum albumin (BSA). When HL-60 cells were induced to differentiate along the granulocytic pathway by incubation with dimethylsulfoxide (DMSO), attachment to hemonectin was reduced. Thus, both primary BM granulocytic cells and a granulocytic cell line show preferential attachment of those cells at earlier stages of differentiation. This developmentally regulated binding suggests a mechanism for release of maturing BM into the peripheral circulation.

Adolescent↗
PubMed1990

Thrombospondin functions as a cytoadhesion molecule for human hematopoietic progenitor cells.

We explored the role that thrombospondin (TSP), a multifunctional extracellular matrix protein, plays in hematopoietic cell-cell and cell-matrix interactions. Thrombospondin synthesis is differentially regulated in human long-term bone marrow cultures. Consistent with this, human hematopoietic progenitor cells of all three lineages (erythrocyte, megakaryocyte, and granulocyte) use TSP as an attachment protein. However, terminally differentiated cells (erythrocytes and neutrophils) show absent or reduced attachment to TSP. The region within the TSP molecule that mediates cell attachment (cell binding domain) was delineated by examining both attachment to proteolytic fragments of TSP and by inhibition of cytoadhesion using monoclonal antibodies directed against TSP domains. The cell binding domain resides toward the C-terminus of a 140 Kd chymotryptic fragment of TSP. We conclude that thrombospondin functions as a hematopoietic cytoadhesion molecule, capable of binding primary hematopoietic progenitor cells, and may, therefore, be important in blood cell development.

Binding Sites↗
PubMed1990

Fetal expression of hemonectin: an extracellular matrix hematopoietic cytoadhesion molecule.

Hemonectin, a component of bone marrow extracellular matrix, is a lineage- and organ-specific attachment molecule for cells of the granulocytic lineage. We hypothesized that hemonectin is an important marker of fetal granulopoiesis that is developmentally regulated during the ontogeny of the hematopoietic system. Murine hematopoiesis originates in the yolk sac and subsequently appears in the liver, spleen, and bone marrow. Using an affinity-purified polyclonal antibody to purified hemonectin as a probe of developing hematopoietic organs, we observe that hemonectin is coordinately expressed at developmental stages of the mouse in those tissues that are supporting hematopoiesis. Multiparameter flow cytometric analysis reveals that approximately 7% of fetal liver cells express hemonectin by day 13 of gestation, and that 32% of the cells are positive by day 19. Additionally, restricted hemonectin expression is noted in other tissues (cartilage, skin, developing bone, and capillary endothelial cells), suggesting that this molecule subserves other developmental functions and/or belongs to a previously unrecognized family of molecules.

Animals↗
PubMed1990

Regulation of megakaryocyte phenotype in human erythroleukemia cells.

Induction of human erythroleukemia (HEL) cells with nanomolar tumor-promoting phorbol myristate acetate (PMA) diesters results in the synchronous acquisition of multiple markers of the megakaryocyte phenotype. Induced cells markedly increase their content of cytoplasm and show features of morphological maturation. At the ultrastructural level, PMA-treated cells show increases in cytoplasm, nuclear lobulation and nucleolar content, and free ribosomes. Limited numbers of cells also express alpha-granules and nascent demarcation membrane systems. Functionally, PMA-stimulated HEL cells express increased amounts of the megakaryocyte/platelet proteins: glycoprotein IIb/IIIa, platelet factor 4, von Willebrand factor, glycoprotein Ib, and thrombospondin. No changes are observed in antigenic markers of the erythroid (glycophorin A) or macrophage lineages (MO-1 or MO-2). The increases in antigenic expression are rapid, reaching maximum levels within 3-4 d under serum-free conditions. Treatment with PMA also abruptly (within 1-2 d) inhibits cellular division in these cells. Washout studies indicate that phorbols exert their effect within 18-24 h, the approximate cell cycle time for these cells. Consistent with proliferative arrest, c-myc proto-oncogene transcripts begin to decline within 8 h of PMA treatment, although transcripts of c-myb are unaffected. Importantly, megakaryocyte differentiation is associated with endomitotic DNA synthesis (i.e., continued DNA synthesis in the absence of mitosis and cytokinesis), with HEL cells reaching a DNA content of 3-12 times that of unstimulated cells. Endomitosis is coordinately regulated with changes in antigenic expression and cell size such that those cells having the highest DNA content are the largest and also express the greatest levels of antigen.

Antigens↗
PubMed1990

Expression of human bone-related proteins in the hematopoietic microenvironment.

Given the intimate relationship between bone and bone marrow, we hypothesized that the human bone marrow may function as a source (or reservoir) of bone-forming progenitor cells. We observed a population of cells within the bone marrow which produce bone-specific or bone-related proteins. The production of these proteins was developmentally regulated in human long-term bone marrow cell cultures; the bone protein-producing cells (BPPC) are observed under serum-free, short-term culture conditions, respond to bone-related and not hematopoietic growth factors, and are derived from a population of low-density, nonadherent, My10-negative (or low My10 density), marrow cells (My10 is an antigen found on most hematopoietic progenitor cells). Cultivation of marrow-derived BPPC in secondary, serum-containing cultures results in their differentiation into osteoblastlike cells. At this stage of development, BPPC produce an extracellular matrix which incorporates both bone-related proteins and radiolabeled calcium. Human bone marrow BPPC thus represent a newly described cell phenotype important to both bone and hematopoietic cell biology.

Antigens, Neoplasm↗
PubMed1989

Signal transduction events in in vitro megakaryocytopoiesis.

Understanding of the events following factor-mediated megakaryocyte development is hindered by a lack of adequate quantities of purified progenitor cells or progenitor cell lines. In order to study the intracellular processes activated during development, probes of various signal transduction systems are used to perturb megakaryocyte colony formation. Studies utilizing tumor promoting phorbol diesters and calcium ionophores show that protein kinase C and calcium mobilization (and/or influx) are activated during megakaryocyte development. Examination of the adenylate cyclase complex, with agonists such as cholera toxin etc., implicate cyclic AMP as another mediator of growth-factor responsiveness. Finally, synergistic interactions occur between the calcium-protein kinase C system and the adenylate cyclase complex. These observations corroborate the multifactorial regulation of megakaryocytopoiesis postulated by Williams and coworkers. The data further provide an intracellular mechanism(s) by which megakaryocytic growth factors regulate cellular development.

Animals↗
PubMed1988

Human recombinant granulocyte-macrophage colony stimulating factor and interleukin 3 have overlapping but distinct hematopoietic activities.

The hematopoietic stimulatory activities of human recombinant IL-3 and granulocyte-macrophage colony stimulating factor (GM-CSF) were directly compared using highly enriched human bone marrow progenitor target cells. IL-3 supported a larger number of erythroid and megakaryocytic progenitor cells than did GM-CSF, while GM-CSF supported more myeloid progenitors. IL-3 directly stimulated the division and migration of primitive erythroid burst forming units, while GM-CSF merely sustained their net survival in culture without promoting division and expansion. IL-3 promoted the formation of larger numbers of multipotential granulocyte-erythroid-macrophage-megakaryocyte colony forming unit--derived colonies than did GM-CSF. These data indicate that human IL-3 and GM-CSF have overlapping but distinct hematopoietic activities, and suggest a potential role for the clinical application of combined IL-3/GM-CSF therapy.

Adult↗
PubMed1988

Synergistic regulation of human megakaryocyte development.

Little information exists concerning differing levels of regulation occurring during human megakaryocyte development. We hypothesize that megakaryocytic proliferation and maturation is controlled by two, synergistic regulatory factors. One, megakaryocyte colony-stimulating activity, is an obligate requirement for colony formation and drives the development of relatively immature cells. Megakaryocyte colony-stimulating activity is a functional component of the human recombinant proteins, interleukin 3 or GM-CSF. Human recombinant growth factors, interleukin 1, interleukin 6, or crythropoietin, do not effect megakaryocyte development either alone or in combination with interleukin 3. Full maturation requires a second synergistic activity which increases megakaryocyte number, size, and cytoplasmic and antigenic content. In culture, this synergistic regulator augments maturation by increasing the number of colonies, colony cellularity, and size. In suspension cultures, this cofactor increases megakaryocyte cytoplasmic and antigenic content, and shifts the morphological distribution from immature to mature megakaryocytes. Finally, this activity also increases the number of antigen positive megakaryocytes, either by stimulating proliferation or conversion of antigen-negative to antigen-positive cells. Comparative studies of megakaryocytic regulation suggests that this in vitro regulator mimicks some of the known effects of thrombopoietin in vivo.

Adolescent↗
PubMed1988

Cholera toxin and phorbol diesters synergistically modulate murine hematopoietic progenitor cell proliferation.

Little information exists concerning the role of guanine nucleotide-binding proteins (GNBP) in hematopoietic progenitor cell proliferation. We hypothesized that GNBP-mediated activation of adenylate cyclase plays an important role in factor-driven hematopoietic cell proliferation. Using cholera toxin and other probes of the cyclase system, we observe that cyclase activation results in a lineage-specific amplification of megakaryocytic progenitor cell numbers, an intermediate effect on erythroid progenitors, and, conversely, an inhibition of granulocytic colony formation. The effect of GNBP activation is synergistic with, and dependent upon, concomitant activation of the cells with phorbol diesters. This suggests a role for both calcium-dependent mechanisms, such as protein kinase C activation, and for other processes mediated by GNBP and cyclic AMP. The use of an intracellular calcium antagonist (Quin-2) partially abrogates the GNBP-mediated response, confirming that the effects of GNBP activation involve both calcium-dependent and calcium-independent processes. We conclude that hematopoietic progenitor cells are influenced by lineage-specific alterations in GTP-binding protein function, which affects both adenylate cyclase activity and calcium homeostasis.

Animals↗
PubMed1988

Detection of human megakaryocyte antigens by solid-phase radioimmunoassay.

Many studies report the presence of platelet-megakaryocyte-associated antigens on human immature and mature megakaryocytes as well as progenitor cells of this lineage. However, studies of purified megakaryocytes or megakaryocyte colonies yield little information on the effect(s) of growth factors in regulating antigenic expression, the kinetics of expression, or the relative content of antigens expressed. We report the detection of human megakaryocyte antigens using a solid-phase radioimmunoassay. The assay is linear, specific, and detects a range of approximately 300-10,000 megakaryocytes per aliquot. Analyses of unstimulated and stimulated suspension cultures indicate that activities known to influence megakaryocyte development in vitro also increase total megakaryocyte antigenic content. Additionally, studies on human erythroleukemia cells show that those cells constitutively express megakaryocyte antigens and increase these antigens when stimulated. Finally, since the specificity of the assay is that of the primary antibody, this procedure is easily extended to analysis of antigenic expression in other lineages.

Antigens, Surface↗