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T Hoang

Publications and source records attributed to T Hoang.

At least 55 records · Page 3Linked to original sources
PubMed1992

Use of Marfey's reagent to quantitate racemization upon anchoring of amino acids to solid supports for peptide synthesis.

A chromatographic assay has been developed to quantitate racemization occurring during attachment of protected amino acids to peptide synthesis resins. Acidolytic cleavage of deprotected amino acids from supports and subsequent derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey's reagent) gave diastereomers separable by reverse-phase HPLC using aqueous acetonitrile. The assay is reliable to 0.1% racemate with a detection limit of approximately 100 pmol. The technique was applied to several acid-labile resins and was used to evaluate the racemizing characteristics of various anchoring methods.

Alanine↗
PubMed1991

Interferon-gamma enhances growth factor-dependent proliferation of clonogenic cells in acute myeloblastic leukemia.

Interferon-gamma (IFN-gamma) has been reported to antagonize the stimulatory effect of various conditioned media on the growth of normal hematopoietic progenitor cells and clonogenic blasts from patients with chronic myelogenous leukemia (CML) and acute myeloblastic leukemia (AML). In the present study, using purified recombinant cytokines and homogenous cell populations, we provide evidence for a synergistic or additive effect of IFN-gamma with recombinant human (rhu) hematopoietic growth factors in the stimulation of clonogenic blasts from most AML patients examined. Under conditions of limiting cell concentration, rhuIFN-gamma alone showed little effect on blast proliferation, whereas in conjunction with recombinant human interleukin-3 (rhuIL-3), IFN-gamma significantly enhanced colony formation in 13 of 15 AML cases. Maximal stimulation was obtained at low concentrations of IFN-gamma (2 to 20 pmol/L) in four cases and at higher concentrations (700 to 7,000 pmol/L) in the remainder. IFN-gamma also synergized with recombinant human granulocyte-macrophage colony-stimulating factor (rhuGM-CSF) in 9 of 13 cases. Within 1 hour of exposure, IFN-gamma induced a twofold to fourfold accumulation of tumor necrosis factor alpha (TNF alpha)-specific transcripts in AML blasts and several AML cell lines that include HL-60 and OCI-AML 1. Further, the synergy between IFN-gamma and IL-3 on AML blasts was partially or completely abrogated by a TNF alpha neutralizing antibody, suggesting that growth enhancement by IFN-gamma may be mediated through TNF alpha production in AML blast culture. Exposure of normal precursors (burst-forming unit-erythroid [BFU-E] and colony-forming unit granulocyte-macrophage [CFU-GM]) to IFN-gamma also resulted in significant growth enhancement, suggesting that the proliferative response elicited by IFN-gamma was not limited to AML blasts. Finally, in M07-E, an IL-3-dependent human "megakaryoblastic" cell line, IFN-gamma also significantly enhanced IL-3-supported colony formation, much in the same way as in primary AML blasts. In contrast, IFN-gamma inhibited growth of all CSF-independent leukemic cell lines tested. This inhibition was partially alleviated by anti-TNF alpha antibody. In summary, our data indicate that IFN-gamma can enhance or antagonize cell proliferation, depending on the cell type. Further, TNF alpha appears to mediate the biologic effect of IFN-gamma either in growth stimulation or growth inhibition.

Antibodies↗
PubMed1991

CD66 identifies a neutrophil-specific epitope within the hematopoietic system that is expressed by members of the carcinoembryonic antigen family of adhesion molecules.

Preliminary results from the IVth Leucocyte Culture Conference have classified the monoclonal antibody (MoAb), YTH 71.3.2, as CD66. Two other MoAbs, YPC 2/12.1 and CE6/2D3.1, share a common cellular specificity, reacting with cells of the neutrophil series and colonic epithelium. The YTH 71.3.2 and CE6/2D3.1 MoAbs both recognize a similar CD66 defined epitope that is distinct from that identified by YPC 2/12.1. By Western blotting, these antibodies react with different molecular species from cells of different lineages. The antibodies identify 50- to 55-Kd, 80- to 100-Kd, and 130- to 200-Kd components present in a semi-purified carcinoembryonic antigen (CEA) preparation from colonic adenocarcinomas and a 90- to 130-Kd molecule from HL-60 cells. With the colonic cell line, LS174T, YPC2/12.1 stains diffuse bands of 160 to 200 Kd and 90 to 130 Kd with equal intensity, whereas the binding of CE6/2D3.1 and YTH 71.3.2 is biased toward the lower molecular weight set of molecules. Remarkably, all three antibodies recognize CEA-related molecules. Defined analyses using HeLa cells transfected with CEA, NCA(NCA-50/90), and CGM6(NCA-95) cDNAs show that the three MoAbs identify CEA to varying degrees. While YTH 71.3.2 and CE6/2D3.1 also bind to NCA-50/90, YPC 2/12.1 recognizes an epitope expressed by both the NCA-50/90 and NCA-95 molecular species.

Antibodies, Monoclonal↗
PubMed1991

Interleukin-6 production by the blast cells of acute myeloblastic leukemia: regulation by endogenous interleukin-1 and biological implications.

Coordinate production of interleukin-1 beta (IL-1 beta) and granulocyte macrophage-colony stimulating factor (GM-CSF) or IL-6 by the blast cells of acute myeloblastic leukemia (AML) and normal peripheral blood leukocytes have been previously reported (van der Shoot et al.: Blood 74:2081-2087, 1989; Bradbury et al.: Leukemia 4:44-47 1990a, British Journal of Haematology 16:(in press), 1990b; Rodriguez-Cimadevilla et al.: Blood 76:1481-1489, 1990; Schindler et al.: Blood 75:40-47, 1990). In the present study, we show that IL-6 production by AML blasts is up-regulated by endogenously produced IL-1 beta. Neutralization of the endogenous source of IL-1 results in a significant decrease in IL-6 production, as determined by ELISA. Conversely, exposure of AML blasts to IL-1 alpha results in a significant increase in IL-6 production in 10 of 16 patient samples. Antibodies against IL-1 alpha and -beta also cause a drastic decrease in IL-6 and GM-CSF gene expression by the cells, suggesting that cytokine gene expression in AML blasts is driven, at least in part, by endogenous IL-1. The biologic significance of IL-6 production in culture of AML blasts has been addressed using a neutralizing antibody against IL-6. Our data indicate that IL-6 is important for the survival of clonogenic blasts in culture. In contrast, the survival of the total population of blasts is IL-6-independent, as assessed by the integrity of cellular DNA, even in the presence of anti-IL-6. These observations are consistent with the view that AML blasts might be organized as a lineage, with comparable hierarchy as in normal hemopoiesis and, perhaps, increased heterogeneity despite a homogenous appearance (McCulloch and Till: Blood Cells 7:63-77, 1981; Buick and McCulloch: Control of Animal Cell Proliferation. Academic Press, New York, vol. 1, pp. 25-57, 1985). Buick and McCulloch have identified a subpopulation of AML clonogenic cells with stem-cell-like properties, and suggested that the majority of blasts may have undergone a determination-like step. Our data indicate a marked difference in IL-6 requirement for cell survival between precursors and the majority of blasts, suggesting that IL-6 responsiveness may decrease following a determination-like event, i.e., the reduction in proliferative capacity.

Antibodies↗
PubMed1990

Coordinate secretion of interleukin-1 beta and granulocyte-macrophage colony-stimulating factor by the blast cells of acute myeloblastic leukemia: role of interleukin-1 as an endogenous inducer.

Acute myeloblastic leukemia (AML) blasts have been shown to produce a variety of cytokines in culture such as interleukin-1 (IL-1), IL-6, granulocyte-, macrophage-, and granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF alpha). Using two sensitive and specific enzyme-linked immunosorbent assays for IL-1 beta and GM-CSF, we document in the present study that the production of the two cytokines by AML blasts in culture is coordinated. First, we observe a striking correlation between the levels of GM-CSF and IL-1 beta released by the cells. Thus, a high production of IL-1 beta is always concordant with a high production of GM-CSF and, conversely, low production of IL-1 beta is concordant with low levels of GM-CSF. Second, neutralization of intrinsic IL-1 using antibodies that are specific for IL-1 alpha and -1 beta suppresses the release of GM-CSF by the cells. Third, neutralization of the endogenous source of IL-1 also results in an abrogation of GM-CSF mRNA. Fourth, the production of both IL-1 beta and GM-CSF is up-regulated by exposing AML blasts to an exogenous source of IL-1, suggesting a positive regulation of autocrine growth factor production. Taken together, our results indicate that GM-CSF production by AML blasts is mediated by endogenously produced IL-1. Both IL-1 beta and -1 alpha are produced by AML blasts, although IL-1 beta appears to be more abundant. Spontaneous colony formation by AML blasts is abrogated by the addition of neutralizing antibodies against IL-1 beta and GM-CSF, whereas each antibody alone has little effect on blast proliferation. Taken together, our results are consistent with the view that the production of IL-1 beta by AML blasts supports autocrine growth in culture, through induction of CSFs or other cytokines that stimulate blast proliferation.

Antibodies↗
PubMed1990

Characterization of granulocyte-macrophage colony-stimulating factor receptor on the blast cells of acute myeloblastic leukemia.

Iodinated granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to document the specific binding of GM-CSF to all acute myeloblastic leukemia (AML) samples examined in the present study. There was some heterogeneity in the number of GM-CSF binding sites per cell. To determine whether the low level of binding to some patient samples may be attributed to receptor occupancy by an endogenous source of GM-CSF, we devised an acid wash procedure that could remove surface-bound GM-CSF without affecting receptor properties. We thus document that GM-CSF specific binding to AML blasts before or after acid wash was the same, indicating that the observed heterogeneity in binding is not the result of receptor occupancy by an endogeneous source of GM-CSF. Saturation analyses are in favor of the presence of two classes of binding sites on AML blasts: a high-affinity receptor that binds GM-CSF with a dissociation constant (kd) of 3 to 73 pmol/L and a second class of low-affinity receptor that binds GM-CSF with a kd of 1 to 10 nmol/L. Binding studies with two established cell lines KG-1, and IRCM-8 also showed the presence of two classes of binding sites with high and low affinities. Analysis of GM-CSF titration curves in culture indicate that the median effective concentration required for stimulation of blast colony formation (EC50 = 5-36 pmol/L) were in the range of the kd of the high-affinity binding site, suggesting that this high-affinity binding site mediates the proliferative response.

Cell Division↗
PubMed1990

Protein phosphorylation in developing and regenerating rat kidney.

Renal cytosolic extracts from rats of different ages and mononephrectomized rats were incubated with gamma-[32P]ATP and analysed by high resolution two-dimensional electrophoresis and autoradiography. Extracts from new-born and young rats showed a great number of phosphorylated proteins migrating between the origin and Mr 52,000. Among these proteins, the group co-migrating with phosphorylase b (Mr 97,000) was particularly evident in new-born and days-old rats. In extracts from mature rats, other proteins of lower molecular weight, particularly those migrating between Mr 60,000 and 44,000, became intensely phosphorylated. The number and intensity of phosphorylated proteins from extracts of normal and nephrectomized rats, however, did not vary. Activity of cAMP-dependent protein kinase and [3H]cAMP binding was also modified during neonatal development but not in compensatory renal growth. Since cAMP-PK and protein phosphorylation are known to be regulated in response to hormonal stimulations, these results may provide good indications for the understanding of hormonal involvement in kidney growth.

Animals↗
PubMed1990

IL-3 inhibits the binding of GM-CSF to AML blasts, but the two cytokines act synergistically in supporting blast proliferation.

Equilibrium binding of 125I-labeled recombinant granulocyte macrophage colony-stimulating factor (GM-CSF) to the blast cells of acute myeloblastic leukemia (AML) revealed the presence of two classes of binding components of high and low affinity, with dissociation constants (Kd) in the range of 5-10 pM and 1-10 nM, respectively. Specificity studies revealed that interleukin-3 (IL-3) could partially inhibit the binding of GM-CSF to AML blasts and to the cells of the leukemic lines M07-E, KG-1, and HL-60. The inhibition of GM-CSF binding by IL-3 was directly dependent on the presence of IL-3 receptors. Analysis of competition curves indicated that the Kd and the number of binding sites per cell of unlabeled and iodinated GM-CSF were identical. In contrast, the inhibition of GM-CSF binding by IL-3 was mediated by IL-3 occupancy of a high affinity receptor only, with the same number of sites as the high affinity GM-CSF receptor but a slightly higher Kd. Despite this competitive binding, IL-3 augmented AML blast proliferation in the presence of GM-CSF, indicating that the two growth factors have converging pathways in supporting blast proliferation. In striking contrast to AML blasts, GM-CSF binding to neutrophils was compatible with the presence of only one class of binding site of intermediate affinity (Kd approximately 100-160 pM). Furthermore, IL-3 does not compete for the binding of GM-CSF to neutrophils.

Binding Sites↗
PubMed1989

Tumor necrosis factor alpha stimulates the growth of the clonogenic cells of acute myeloblastic leukemia in synergy with granulocyte/macrophage colony-stimulating factor.

TNF-alpha has been shown to antagonize the proliferative effects of growth factors present in crude conditioned media from PHA-stimulated leukocytes or cell lines on the clonogenic cells of acute myeloblastic leukemia (AML) (19,21). In the present study, we investigated the responses of AML blasts to TNF-alpha in the presence of defined growth factors (recombinant granulocyte/macrophage-CSF [rGM-CSF], recombinant granulocyte-CSF [rG-CSF], rIL-3, and rIL-1) and under conditions described for autocrine stimulation (32). While TNF-alpha antagonized the stimulatory effects of G-CSF and IL-3 on blast progenitors, TNF-alpha did not affect blast colony formation in the presence of IL-1. Unexpectedly, TNF-alpha significantly enhanced blast proliferation in the presence of GM-CSF. Further, TNF-alpha also acted synergistically with an endogenous source of growth stimulatory signal to promote proliferation of blast clonogenic cells. Thus, on human leukemic cells, TNF-alpha appears to be a molecule that is at least bifunctional, having the ability to either support or inhibit cell proliferation, depending on the other growth factors present. It is postulated that the proliferative response of blast progenitors to TNF-alpha under conditions that favor autocrine stimulation may represent one property that allows the cells to escape from negative regulation and proliferate in AML.

Blotting, Northern↗
PubMed1989

Transgenic mice carrying the mouse mammary tumor virus ras fusion gene: distinct effects in various tissues.

Transgenic mice carrying the v-Ha-ras oncogene under the control of the mouse mammary tumor virus long terminal repeat were produced. These mice exhibit several phenotypes: mammary tumors, bilateral hyperplasia of the harderian lacrimal gland, primary bronchio-alveolar lung adenocarcinoma, and splenomegaly. High levels of the transgene RNA were detected in mammary, harderian, and lung tumors. Accumulation of cells of the myeloid lineages was found in enlarged spleens. This phenotype may represent an indirect effect of v-Ha-ras expression on myeloid progenitors. Our data illustrate the cell-specific effects of v-Ha-ras.

Animals↗
PubMed1988

Interleukin 1 enhances growth factor-dependent proliferation of the clonogenic cells in acute myeloblastic leukemia and of normal human primitive hemopoietic precursors.

IL-1 is released by activated monocytes and is thought to be a key mediator of the host immune response. The availability of the purified and, more recently, recombinant IL-1 has allowed the characterization of other biological properties of this molecule. Thus, IL-1 is thought to have the same properties as hemopoietic 1, a growth factor that has been shown to act on primitive murine hemopoietic cells. Here we report that rIL-1 acts synergistically with granulocyte/macrophage CSF (GM-CSF) or granulocyte CSF in the stimulation of clonogenic cells from many patients with acute myeloblastic leukemia (AML). Although IL-1 by itself has no effect on AML blasts, it can support colony formation under conditions where there is detectable production of endogenous GM-CSF. IL-1 also promotes the growth of multipotential progenitors from normal human bone marrow cells in the presence of GM-CSF. These observations support the hypothesis that in the hemopoietic system, IL-1 has a selective effect on primitive precursors.

Bone Marrow Cells↗
PubMed1988

Segregation of precursors with high proliferative potential from their differentiated progeny in a myeloma cell line.

Human myeloma colonies were grown from the peripheral blood of a patient with multiple myeloma who was unresponsive to any further therapy. The majority of primary myeloma colonies contained cells that were able to form secondary colonies upon replating and facilitated the establishment of a myeloma cell line (OCI-My5). The recloning procedure was repeated for colonies that contained the highest and lowest number of clonogenic progenitors. This approach allowed the selection of clonogenic cells with high self-renewal potential. Using a cell separation procedure based on differences in sedimentation velocity, clonogenic myeloma cells with high self-renewal were found to be smaller than the majority of clonogenic myeloma cells. Furthermore, cells with increasing size did not only display reduced self-renewal ability but showed significant increases in the content of mRNA transcripts for lambda light chains. These data were consistent with the view that clonogenic myeloma cells are heterogeneous with respect to self-replication and that they give rise to more differentiated progeny, as documented by increasing mRNA levels for the M-protein.

Cell Differentiation↗
PubMed1988

Interleukin-6 enhances growth factor-dependent proliferation of the blast cells of acute myeloblastic leukemia.

The effects of recombinant interleukin-6 (IL-6) on the proliferation of blast precursors present in the peripheral blood of patients with acute myeloblastic leukemia (AML) was investigated. IL-6 had little effect by itself; however, it synergized with granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) in the stimulation of AML blast colony formation. Responsiveness of blast progenitors to IL-6 was heterogeneous. On normal bone marrow cells the same synergy was observed on granulocyte and monocyte precursors (GM-CFC), while there was no significant effect on erythroid and multipotential precursors.

Cell Division↗
PubMed1988

Transforming growth factor beta inhibits the proliferation of the blast cells of acute myeloblastic leukemia.

The effect of transforming growth factor beta (TGF beta) on proliferation and differentiation of peripheral blast precursors in acute myeloblastic leukemia (AML) was investigated. TGF beta induced a dose-dependent inhibition of blast clonogenic cells in suspension and methylcellulose cultures in the presence of optimal concentrations of stimulators provided by conditioned media from the bladder cell line HTB9 (HTB9-CM) or the recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF). On removal of TGF beta, blast clonogenic cell proliferation recovers to the same level as that observed in control cultures, indicating that the effect is reversible. There was no induction of cell differentiation, as indicated by morphological and functional studies (production of superoxyde anions). Cell cycle analysis by thymidine uptake and flow cytometry with a DNA binding dye indicated that TGF beta caused a delay in progression into S and G2/M phases of the cell cycle without affecting cell viability. Thus, TGF beta appears to have a cytostatic rather than cytolytic effect on blast precursors and might therefore play a role as a negative regulator in hematopoiesis.

Antineoplastic Agents↗