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J Hatzfeld

Publications and source records attributed to J Hatzfeld.

At least 37 records · Page 2Linked to original sources
PubMed1993

Co-stimulatory effects of steel factor, the c-kit ligand, on purified human hematopoietic progenitors in low cell density culture.

Steel factor (SF), the ligand for the c-kit, also called kit ligand, stem cell factor, or mast cell growth factor, was evaluated on colony formation alone or in combination with other cytokines, from purified human hematopoietic CD34+ cells in low density cell culture. SF alone had a slight effect on granulocyte (G) and macrophage (M) colony formation. It synergized with other cytokines on colony formation from colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM), erythroid and granulocyte-macrophage (CFU-GM) progenitors. However, combination of SF with lineage-specific factors, such as erythropoietin (Epo) or/and granulocyte colony-stimulating factor (G-CSF) was not sufficient for the proliferation of multipotential progenitors (CFU-GEMM). These multipotential progenitors required the presence of multi-lineage factors, such as interleukin 3 (IL3) or granulocytic-macrophage CSF(GM-CSF) for their development.

Cell Count↗
PubMed1992

Cryopreservative effect of leupeptin on early human bone marrow progenitors.

Leupeptin, a thiol- and serine-proteinase inhibitor of low molecular weight, quickly enters viable cells. This property has been used to protect cells during thawing against intracellular proteolytic activities released by injured lysosomes. The bone marrow nucleated cells were frozen without rate-controlled freezing devices. Concentrations ranging from 0.1 to 1 microM of leupeptin allow to recover 87% of the most immature multipotent bone marrow progenitors which can develop in vitro into large multilineage colonies, instead of 58% recovery without leupeptin. The protective effect of leupeptin is particularly useful to freeze cells difficult to cryopreserve or when freezing-control equipment are not available.

Bone Marrow↗
PubMed1992

c-fos mRNA constitutive expression by mature human megakaryocytes.

By in situ hybridization with a c-fos probe, we have shown that human bone marrow megakaryocytes cultured in the presence of 20% aplastic anemia plasma constitutively express c-fos mRNA. At day 0, megakaryocytes are mostly immature and only 3% of them are labeled. The number of labeled cells reached 23% after 12 days of culture. Interleukin 3 (IL-3) and IL-6 added together at day 10 further increased this number to 31% 2 days later. Mature labeled megakaryocytes were more numerous and more strongly labeled than immature ones. These results suggest that c-fos could play a role in megakaryocytic terminal differentiation, either in the polyploidization or in the thrombopoietic function unique to these cells.

Genes, fos↗
PubMed1991

Release of early human hematopoietic progenitors from quiescence by antisense transforming growth factor beta 1 or Rb oligonucleotides.

We have used antisense oligonucleotides to study the roles of transforming growth factor beta (TGF-beta) and the two antioncogenes, retinoblastoma susceptibility (Rb) and p53, in the negative regulation of proliferation of early hematopoietic cells in culture. The antisense TGF-beta sequence significantly enhanced the frequency of colony formation by multi-lineage, early erythroid, and granulomonocytic progenitors, but did not affect colony formation by late progenitors. Single cell culture and limiting dilution analysis indicated that autocrine TGF-beta is produced by a subpopulation of early progenitors. Antisense Rb but not antisense p53 yielded similar results in releasing multipotential progenitors (colony-forming unit-granulocyte/erythroid/macrophage/megakaryocyte) from quiescence. Rb antisense could partially reverse the inhibitory effect of exogenous TGF-beta. Anti-TGF-beta blocking antibodies, antisense TGF-beta, or Rb oligonucleotides all had similar effects. No additive effects were observed when these reagents were combined, suggesting a common pathway of action. Our results are consistent with the model that autocrine production of TGF-beta negatively regulates the cycling status of early hematopoietic progenitors through interaction with the Rb gene product.

Antigens, CD↗
PubMed1991

Mitogenic properties of major extracellular proteins.

The major plasma and extracellular matrix proteins are multifunctional molecules. Some, such as fibrinogen or C3, have one domain that binds adhesion receptors and another that specifically binds and activates a separate, mitogenic receptor. In this review, Jean-Pierre Lévesque, Antoinette Hatzfeld and Jacques Hatzfeld describe adhesion and mitogenic receptors that bind to distinct domains of the same extracellular matrix protein and discuss the possibility of common ancestral genes for cell adhesion molecules, extracellular matrix proteins, integrins, immunoglobulins, growth factors and their receptors.

Complement C3↗
PubMed1991

DNA transfection in COS cells: a low-cost serum-free method compared to lipofection.

We describe a defined medium that allows efficient DNA transfections in COS cells and transient expression of the corresponding recombinant protein in serum-free conditions. With a modified DEAE-dextran/chloroquine method, we obtained 80% more transfected cells expressing the recombinant human interleukin-2 receptor than with transfection with cationic liposomes, one of the most efficient techniques to date. The absence of serum in the culture medium should reduce subsequent purification steps for production of recombinant mammalian proteins. Moreover, it should allow investigations dealing with the role of serum or other exogenous factors on mRNA stability or post-translation events during protein synthesis.

Cell Line↗
PubMed1990

A mitogenic fibrinogen receptor that differs from glycoprotein IIb-IIIa. Identification by affinity chromatography and by covalent cross-linking.

The mitogenic effect of human fibrinogen on the hemopoietic cell lines Raji and JM is mediated by a specific receptor with biochemical and functional properties different from those of the platelet fibrinogen receptor, the glycoprotein complex IIb-IIIa (Levesque, J.P., Hatzfeld, A., and Hatzfeld, J. (1986) Proc. Natl. Acad. Sci. U.S. A. 83, 6494-6498; Levesque, J.P., Hatzfeld, A., Hudry-Clergeon, G., Wilner, G.D., and Hatzfeld, J. (1987) J. Cell. Physiol. 232, 303-310). This work describes the identification of the mitogenic fibrinogen receptor (MFR) by two independent methods, affinity chromatography and covalent cross-linking. Affinity chromatography of surface-labeled cell extracts on fibrinogen-Sepharose revealed a 94 +/- 2-kDa membrane protein that bound specifically to fibrinogen-Sepharose only on cells that expressed the MFR. Its molecular mass was not modified after reduction. This was confirmed by cross-linking fibrinogen to surface-labeled Raji cells using the cleavable cross-linkers, ethyleneglycobis(succinimidyl succinate) and dithiobis(succinimidyl propionate). Complexes between fibrinogen and iodinated cell membrane proteins were immunoprecipitated by anti-fibrinogen antibodies. The biochemical cleavage of these immunoprecipitated conjugates gave rise to a 92 +/- 3-kDa membrane protein whose molecular mass was not modified after reduction. We conclude that fibrinogen binds specifically to a 92-94-kDa MFR which does not belong to the integrin family.

Cell Line↗
PubMed1990

The down-regulation of the mitogenic fibrinogen receptor (MFR) in serum-containing medium does not occur in defined medium.

Normal human hemopoietic cells such as early bone marrow progenitors, or lymphoma-derived cell lines such as Raji or JM cells, possess a low-affinity receptor specific for fibrinogen. This receptor triggers a mitogenic effect. It differs from the glycoprotein IIb-IIIa which is involved in fibrinogen-induced platelet aggregation. We demonstrate here that this mitogenic fibrinogen receptor (MFR) can be internalized or reexpressed, depending on culture conditions. Internalization was temperature-dependent. At 37 degrees C in the presence of cycloheximide or actinomycin D, the half-life of cell surface MFRs was 2 h, independent of receptor occupancy. Binding of fibrinogen to the MFR resulted in a down-regulation which was fibrinogen dose-dependent. This occurred in serum-supplemented medium but not in defined medium supplemented with fatty acids. Reexpression of MFRs could be induced in 28 to 42 h by serum removal. The down-regulation of mitogenic receptors in plasma or serum could explain why normal cells do not proliferate in the peripheral blood.

Blood↗
PubMed1988

Binding of C3 and C3dg to the CR2 complement receptor induces growth of an Epstein-Barr virus-positive human B cell line.

The effect of ligand interactions with the C3d/C3dg complement receptor (CR2) on proliferation of human B lymphoblastoid cells was investigated by using cell cultures performed at low density (1 to 1.5 x 10(3) cells/ml) in a serum-free defined medium to which only transferrin had been added. This medium does not allow proliferation of Raji cells which die within 48 hr with formation of polykaryons. Addition of purified human C3 to the cultures resulted in a dose-dependent proliferation of the cells. A steady growth of Raji cells with a doubling time of 36 hr was observed in cultures containing 10 micrograms/ml of C3. A growth rate similar to that observed in the presence of native C3 was found in the presence of equimolar concentrations of purified C3dg but not of C3c. F(ab')2 anti-C3d but not F(ab')2 anti-C3c antibodies inhibited the mitogenic effect of C3. Preincubation of Raji cells with monoclonal antibody OKB7 which directly inhibits the binding of C3dg to CR2, totally suppressed C3-induced growth of the cells. C3 did not enhance growth of the T lymphoma-derived cell line JM and monocytic cell line U937 which do not express CR2. These results provide direct evidence that the interaction between CR2 and C3 fragments stimulates proliferation of human cells of the B lineage. Because CR2 also acts as a receptor for Epstein-Barr virus on B cells, our results may pertain to the B cell mitogenic properties of the virus.

B-Lymphocytes↗
PubMed1987

Evidence for two functionally different fibrinogen receptors on hemopoietic cells: the glycoprotein IIb-IIIa and the mitogenic fibrinogen receptor.

We have previously established that the mitogenic effect of fibrinogen on hemopoietic cell lines Raji and JM is mediated via a specific receptor (Levesque, J.-P. et al.: Proc. Natl. Acad. Sci. USA 83:6494-6498, 1986). In this study, we have further characterized the fibrinogen domain involved in the binding to the mitogenic receptor. This binding was not inhibited either by a monoclonal antibody against the C-terminal sequence of the fibrinogen gamma chains or by synthetic peptides containing the Arg-Gly-Asp sequence. Such inhibition is specific of the platelet fibrinogen receptor, the glycoprotein IIb-IIIa complex. Fragments containing the fibrinogen D domain were the only plasmin degradation products of fibrinogen which were mitogenic. These fragments acted via direct binding on the mitogenic receptor with a Kd of 2.24 X 10(-6) M. This value was similar to the KI value of unlabeled fragments D (2.47 X 10(-6) M). Our results suggest the presence of two different functional types of fibrinogen receptors: the glycoprotein IIb-IIIa receptor responsible both for platelet aggregation and leukocyte adhesion and killing, and the mitogenic receptor involved in proliferation control of hemopoietic cells.

Antibodies, Monoclonal↗
PubMed1986

Dexamethasone-induced stimulation of arachidonic acid release by U937 cells grown in defined medium.

Since the presence of serum in culture media has been shown to alter prostaglandin production, as well as to interfere with the action of anti-inflammatory drugs, we have studied the effect of dexamethasone, a potent steroidal anti-inflammatory drug, on the metabolism of arachidonic acid by human monocyte-like cells (U937) grown in a fully defined medium. Under these culture conditions, dexamethasone (10(-6) M, 24 h) induced a marked stimulation of the release of unmetabolized arachidonic acid into the culture medium. The steroid also induced an inhibition of cell proliferation which became significant only after 48 h of treatment. The accumulation of arachidonic acid in the medium after steroid treatment was associated with a significant inhibition of cell acyltransferase activity, suggesting that steroids may also act upon arachidonic acid metabolism at sites other than those of phospholipase activity.

Acyltransferases↗
PubMed1986

Fibrinogen mitogenic effect on hemopoietic cell lines: control via receptor modulation.

The possibility that the mitogenic effect of fibrinogen, a major plasma protein (3 mg/ml), is mediated by specific membrane receptors was studied. Specific binding analysis showed that fibrinogen receptors are present only on hemopoietic cell lines that respond to its mitogenic effect. The mitogenic fibrinogen receptor is not recognized by antibodies specific for the platelet fibrinogen receptor or is not competitively blocked by synthetic peptides containing the Arg-Gly-Asp sequence, which is common to fibronectin, fibrinogen, vitronectin, and other cell-attachment proteins. The lymphoma-derived pre-B-cells (Raji) have 149,000 receptors, whereas the lymphoma-derived T cells (JM), which are 3 times smaller, have 54,000 receptors. These receptors have a Kd of 2 X 10(-7) M. They are inducible by stimuli specific for the cell lineage: activators of the breakdown of phosphatidylinositol phosphates, such as platelet activating factor for Raji cells, and adenylate cyclase agonists and cAMP analogues for JM cells. The stimuli have no mitogenic effect in the absence of fibrinogen; they do not change the Kd. Each stimulus increases the number of fibrinogen receptors in a dose-dependent manner, which correlates strongly (r = -0.98, n = 5) with an increased growth rate of cells in the presence of fibrinogen. This correlation concludes that the mitogenic effect of fibrinogen is controlled via receptor modulation.

Adenosine Diphosphate↗
PubMed1985

A method to measure receptor binding of ligands with low affinity. Application to plasma proteins binding assay with hemopoietic cells.

A gradient has been developed for separating free ligand from ligand bound to cells growing in suspension. This method can be used with all kinds of ligand but it is particularly useful for those ligands having the tiresome tendency to adhere to the cells non-specifically or to polymerize by themselves. This is the case of fibronectin, fibrinogen, immunoglobulins and many other plasma proteins. The gradient consists of two layers: an upper aqueous phase and a lower hydrophobic organic phase. The aqueous phase, a sucrose buffer, allows the cells to become well dispersed before they enter the hydrophobic phase which excludes the free ligand efficiently. This reduces non-specific binding and allows the accurate measurement of specific binding which could not be obtained with a gradient made of a single phase. Depending upon the size and the density of the cells, and the nature of the ligand, the assay method can be modified by changing the density or the nature of the hydrophilic and hydrophobic phases.

Cell Count↗
PubMed1982

Acetylcholine receptor: effects of proteolysis on receptor metabolism.

Previous studies (Miskin, R., T. G. Easton, and E. Reich, 1970, Cell. 15:1301-1312) have shown that sarcoma virus transformation and tumor promoters reduced the cell surface concentration of acetylcholine receptors (AChR) in differentiating chick embryo myogenic cultures. Both of these agents also induced high rates of plasminogen activator (PA) synthesis in myogenic cultures (Miskin, R., T. G. Easton, A. Maelicke, and E. Reich, 1978, Cell. 15:1287-1300), and the present work was performed to establish whether proteolysis might significantly affect receptor metabolism. Proteolysis in myogenic cultures was modulated by one or more of the following: stimulation of PA synthesis, direct addition of plasmin, removal of plasminogen, or addition of plasmin inhibitors. The results were: (a) When the rates of proteolysis were raised either by addition of plasmin or by stimulating PA synthesis in the presence of plasminogen, both the steady-state concentration and the half-life of surface AChR decreased, but the rate of receptor synthesis was unaffected. (b) The magnitude of these effects, and their dependence on added plasminogen, indicated that proteolysis initiated by plasminogen activation could account almost entirely for the reduction in receptor half-life produced by sarcoma virus transformation and phorbol ester. (c) The rate of receptor synthesis, which is also reduced by viral transformation and tumor promoters, was not modified by proteolysis; hence plasmin action may be responsible for a large part, but not all of the change in surface receptor under these conditions. (d) The plasmin catalysed changes in receptor parameters appear to occur in response to modified membrane metabolism resulting from proteolysis of surface components other than AChR itself.

Animals↗