RESUMEN
A model system for the analysis of intracellular events governing the modification of individual vitamin K-dependent (VKD) proteins by the carboxylase has been developed using recombinant VKD protein-transfected cell lines. When untransfected 293 cells were analyzed by in vitro carboxylation followed by SDS-PAGE, endogenous VKD proteins were not detected. With 293 cells stably-transfected with recombinant native factor IX, most (> 95%) of the carboxylase was in complex with the factor IX, as assayed by adsorption of carboxylase activity to immobilized anti-factor IX antibody. In contrast, with 293 cells stably-transfected with recombinant factor IX deleted in the propeptide sequence (amino acids -18 to -4, delta pro factor IX), no association of factor IX with the carboxylase was observed. This observation was used to specifically isolate and identify the human carboxylase, and carboxylase-associated protein. When the carboxylase was purified from solubilized microsomes from either native factor IX, or delta pro factor IX, stably-transfected 293 cells, a single 98 kDa band was specifically obtained from native factor IX microsomes, but not from delta pro factor IX microsomes. This band was subsequently shown by Western and microsequencing analysis to comprise both the carboxylase and carboxylase-associated protein. This isolation, which represents the first isolation to near homogeneity of both the human carboxylase and the carboxylase from cell lines, will be valuable in isolating enzymatically active recombinant carboxylase, which has been refractile to other purification attempts. This system was also used to show that the human carboxylase in 293 cells is capable of binding and modifying two different liver-derived proteins. Protein C-producing 293 cells were generated from the same 293 progenitor cell line used to created the factor IX-expressing cells. With both factor IX- and protein C-transfected 293 cells, the secreted proteins were almost completely carboxylated, and in microsomes from each cell line the carboxylase was found in near quantitative complex with the two different VKD proteins. Thus the carboxylase modifies both VKD proteins. The approach described here for the analysis of the carboxylase from recombinant VKD protein-transfected cell lines should provide an important new system for studying protein carboxylation and VKD protein-carboxylase interaction.
Asunto(s)
Ligasas de Carbono-Carbono , Factor IX/metabolismo , Ligasas/aislamiento & purificación , Proteínas/aislamiento & purificación , Secuencia de Aminoácidos , Secuencia de Bases , Factor IX/genética , Humanos , Ligasas/genética , Ligasas/metabolismo , Microsomas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Proteína C/metabolismo , Proteínas/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The microsomal gamma-carboxylase catalyzes modification of a limited set of glutamyl residues to gamma-carboxyglutamyl residues in a vitamin K-dependent reaction that also utilizes O2 and CO2. We report the purification to apparent homogeneity of the bovine liver microsomal carboxylase. Affinity chromatography exploiting the association of the carboxylase with prothrombin precursor and carboxylase binding to the propeptide sequence were combined with ion-exchange chromatography and fractionation using immobilized lectins. A 3.5 x 10(5)-fold purification was obtained, which is the highest purification, by a factor of 35, yet reported for this enzyme. A single 98-kDa protein is obtained from this isolation. Carboxylase activity is associated with this protein by two different criteria. Antibodies prepared against the carboxylase detected the 98-kDa protein when used in Western analysis. In addition, the single 98-kDa protein was shown to comigrate with activity when electrophoresed in a nondenaturing gel system. The availability of purified preparations of carboxylase will facilitate an increased understanding of the complex biochemical reaction carried out by this protein.
Asunto(s)
Ligasas de Carbono-Carbono , Ligasas/aislamiento & purificación , Microsomas Hepáticos/enzimología , Animales , Western Blotting , Bovinos , Ligasas/química , Ligasas/inmunología , Peso MolecularRESUMEN
The acute monocytic leukemia cell line, THP-1, is frequently used as a model to study the mechanism of cell-mediated immune response. The model is justified, in part, because THP-1 cells can be induced to express features of activated peripheral blood monocytes or tissue macrophages. The current investigation, however, demonstrates that THP-1 cells differ from normal monocytes in their secretion of interleukin-1 beta (IL-1 beta) following exposure to reagents that induce synthesis. For example, LPS treatment alone did not result in IL-1 beta secretion and very low concentrations were observed when lipopolysaccharide (LPS)-treated cells were simultaneously incubated with silica or silica in combination with hydroxyurea. Silica-enhanced release of IL-1 was related to changes in cell membrane permeability. Recombinant interferon-gamma (rIFN gamma) alone did not induce IL-1 beta secretion and did not significantly increase secretion by LPS- and silica-stimulated cells. In contrast, mezerein stimulation led to higher extracellular concentrations of IL-1 beta and rIFN gamma augmented secretion by mezerein-treated cells. Isoelectrofocusing of conditioned medium and titration of pooled fractions showed a direct correlation between extracellular levels of biologically active and immunoreactive IL-1. The role of IFN gamma in stimulating IL-1 beta secretion was not related to an enhancement of viability or an increase in the proportion of mezerein-treated cells synthesizing DNA. It was concluded that mezerein's regulation of secretion by THP-1 cells depended on the expression of monocyte features, including cell adherence and responsiveness to IFN gamma.
Asunto(s)
Diterpenos , Interleucina-1/metabolismo , Leucemia Monocítica Aguda/metabolismo , Lipopolisacáridos/farmacología , Terpenos/farmacología , Animales , Bioensayo , División Celular , Supervivencia Celular , Ensayo de Inmunoadsorción Enzimática , Humanos , Interferón gamma/farmacología , Interleucina-1/análisis , Interleucina-1/farmacología , Focalización Isoeléctrica , Ratones , Ratones Endogámicos C3H , Proteínas Recombinantes , Timo/citología , Células Tumorales CultivadasRESUMEN
This study examined the secretion of IL-1 alpha and IL-1 beta by THP-1 leukemia cells following activation with mezerein and promotion of synthesis by interferon (IFN-gamma). Interleukin-1 (IL-1) was not detected by co-mitogenic thymocyte assays of crude supernates. Isoelectrofocusing of concentrated medium showed that all biologically active IL-1 migrated at a pH of 6.8-7.2, indicating that the major secreted form was IL-1 beta. Double antibody ELISA confirmed the presence of IL-1 beta, but failed to detect IL-1 alpha in isofocused fractions. Although it appeared that THP-1 cells do not secrete IL-1 alpha; an inhibitor of thymocyte response to IL-1 was present in conditioned medium, migrated in an acidic pH range and masked the expression of biologically active rIL-1 alpha and rIL-1 beta. In contrast, IL-1 alpha was detected using a cell blotting assay. This technique permitted visualization of subpicogram levels of IL-1 when secreted by cells attached to an immunoblotting paper. Cell blotting showed that a greater proportion of attached cells incubated for 24 h in medium containing mezerein and IFN-gamma secreted IL-1 than cells in control medium. In conclusion, the amount of immunoreactive or biologically active IL-1 alpha secreted by stimulated THP-1 cells appeared to be much lower than that reported for human peripheral blood monocytes.
Asunto(s)
Diterpenos , Interleucina-1/metabolismo , Leucemia Monocítica Aguda/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Terpenos/farmacología , Células Tumorales CultivadasRESUMEN
Human recombinant interleukin 1 alpha (IL-1 alpha) and IL-1 beta inhibited the replication of the mammary tumor cell line, MDA-MB-415; stimulated division in the colon carcinoma, SW-48; and had no effect on the growth of the milk mammary line, HBL-100. Inhibition of growth was reflected in a significant decrease in DNA synthesis accompanying a transient increase in RNA synthesis. Specific binding of 125I-labeled recombinant IL-1 beta by MDA-MB-415 and SW-48 reached a maximum by 2 h of incubation, and an equivalent amount was bound by each cell type. Binding was inhibited in a dose-dependent manner by unlabeled IL-1 alpha or IL-1 beta. Scatchard plot analysis revealed that MDA-MB-415 cells expressed approximately 700 binding sites with an apparent dissociation constant of 8.8 x 10(-10) M. Reversibility of growth inhibition was independent of dose or time of incubation, but DNA synthesis did not return to control values. Flow cytometric analysis of DNA content showed that growth inhibition was cell cycle phase nonspecific with a slight reduction in the proportion of cells in S phase. The major conclusion from these studies was that inhibition or stimulation of malignant cell growth by IL-1 was related to the presence of receptor sites.
Asunto(s)
Interleucina-1/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Unión Competitiva , División Celular/efectos de los fármacos , Línea Celular , Neoplasias del Colon/patología , Citometría de Flujo , Humanos , Neoplasias Mamarias Experimentales/patología , Receptores de Interleucina-1RESUMEN
THP-1 is an acute monocytic leukemia cell line which acquires phenotypic and functional monocytoid-like features following incubation with mezerein. The current study concerned the modulation of these features by rIFN gamma. rIFN gamma induces the time-dependent enhancement of HLA-DR expression in the presence or absence of mezerein but has no effect on the expression of Leu-M1, Leu-M2, or Leu-M3 antigens. CSF-1 production following mezerein activation was reduced by incubation in the presence of 10(3) and 10(4) units/ml rIFN gamma. This was confirmed through both biological assays with mouse bone marrow cells and an indirect ELISA. In contrast, the concentration of growth inhibitory activity in conditioned medium was increased by rIFN gamma. A small but significant increase in IL-1 beta concentration in conditioned medium was detected using a sensitive double-antibody ELISA and a radioimmunoassay. The results infer that the functional characteristics of this leukemia cell line are modulated by rIFN gamma in a manner qualitatively similar to that reported for IFN gamma treated normal monocytes.
Asunto(s)
Interferón gamma/farmacología , Leucemia Monocítica Aguda/metabolismo , Antígenos de Superficie/análisis , Factores Estimulantes de Colonias/biosíntesis , Inhibidores de Crecimiento/biosíntesis , Humanos , Interleucina-1/biosíntesis , Leucemia Monocítica Aguda/inmunología , Células Tumorales CultivadasRESUMEN
Polyclonal antisera to human recombinant interleukin-1 beta have been developed in rabbits and mice. When measured by indirect enzyme-linked immunoassays, the antisera bound rIL-1 beta in a dose-dependent manner but did not bind to rIL-1 alpha or murine rIL-1. Both antisera specifically neutralized thymocyte activation induced by rIL-1 beta or normal monocyte IL-1 and growth inhibition induced by rIL-1 beta. The antiserum specificities led us to examine a double antibody ELISA for quantitating IL-1 beta. The linear portion of the standard curve from an ELISA against rIL-1 beta ranged from 0.5 to 10.0 half-maximal units of [3H]thymidine incorporation in mouse thymocyte cultures. A similar correlation was observed using monocyte derived IL-1 from two sources. The major result of this work was the observation that polyclonal antibodies made against a recombinant molecule also recognized natural IL-1. We suggest that these antisera recognize epitopes unique to IL-1 beta which reside at or near regions of the molecule responsible for biological activity.
Asunto(s)
Anticuerpos/inmunología , Interleucina-1/análisis , Secuencia de Aminoácidos , Animales , Ensayo de Inmunoadsorción Enzimática , Humanos , Sueros Inmunes/inmunología , Interleucina-1/inmunología , Ratones , Ratones Endogámicos C3H , Proteínas Recombinantes/análisisRESUMEN
The human monocytic leukemia cell line, THP-1, acquires macrophage-like characteristics following exposure to mezerein. Serum-free medium conditioned by mezerein-activated cells was observed to contain colony-stimulating factor (CSF) activity in assays with murine bone marrow cultures. Isoelectrofocusing revealed that CSF activity displayed charge heterogeneity and migrated in a pl range of 4.4-5.3. Treatment with neuraminidase did not affect biological activity but did reduce charge heterogeneity. Reisofocusing of neuraminidase-treated CSF revealed a peak of activity at pl 4.9. The active component was shown to be an acidic sialoglycoprotein, resistant to proteolytic cleavage but completely inactivated by 2-mercaptoethanol. This CSF has been purified from THP-1-conditioned serum-free medium by preparative isoelectrofocusing, gel filtration through Sephacryl S-200, ion exchange chromatography on DEAE-Sephacel, neuraminidase treatment, and tris-glycinate polyacrylamide gel electrophoresis (PAGE). Elution from SDS-PAGE revealed a single peak of activity corresponding to an apparent molecular weight of 70,000 daltons. Preliminary characterization of the bone marrow cells in colonies showed that THP-1 cells produced macrophage-specific CSF.
Asunto(s)
Factores Estimulantes de Colonias/aislamiento & purificación , Leucemia Mieloide/fisiopatología , Animales , Células de la Médula Ósea , Adhesión Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Factores Estimulantes de Colonias/biosíntesis , Factores Estimulantes de Colonias/farmacología , Replicación del ADN/efectos de los fármacos , Estabilidad de Medicamentos , Humanos , Focalización Isoeléctrica , Ratones , Timidina/metabolismoRESUMEN
After mezerein treatment of suspension cultures of the acute monocytic human leukemia cell line THP-1, cells became adherent to plastic culture surfaces, lost division potential, acquired Fc receptors, displayed phagocytic activity, and expressed increased nonspecific esterase staining. Serum-free RPMI 1640 medium conditioned by adherent THP-1 cells was examined for the presence of biological response modifiers. Preparative isoelectrofocusing of concentrated medium in the presence of various pH gradients of Ampholine ampholytes resulted in the separation of the following activities: fibroblast growth-stimulating activities in pH ranges of 4.10-4.55 and 5.30-5.45; colony-stimulating factor (CSF) for mouse bone marrow cells at pH 4.10-4.55; and a malignant cell growth inhibitor comigrating with a lymphocyte-activating factor (interleukin-1) at pH 6.70-6.95. Both CSF and the fibroblast growth stimulator isofocused at pH 4.10-4.55 coeluted following molecular-sieve chromatography through P-100. CSF-induced colonies were composed of nongranulocytic mononuclear cells. Chromatography through an ACA-54 column separated interleukin-1 from most of the growth-inhibitory activity.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Diterpenos , Sustancias de Crecimiento/metabolismo , Leucemia Mieloide/metabolismo , Terpenos/farmacología , Bioensayo , Adhesión Celular , Línea Celular , Factores Estimulantes de Colonias/metabolismo , Humanos , Interferones/metabolismo , Interleucina-1/análisis , Macrófagos/fisiologíaRESUMEN
Medium conditioned by mezerein-treated human acute monocytic leukemia cells (THP-1) stimulated human fibroblast replication. Maximum mitogenic activity was elaborated by THP-1 cells with a 24-hr incubation in 10(-7) M mezerein (activator phase) followed by a 36-hr incubation in insulin-supplemented serum-free Roswell Park Memorial Institute (RPMI)-1640 medium (effector phase). Growth stimulation was not due to the presence of residual mezerein. We previously reported that leukemia cells also produced a growth inhibitor. Fibroblast stimulation was resolved by isoelectrofocusing into several active fractions separate from the growth inhibitory activity for malignant mammary cells. Conditioned medium was mitogenic for fibroblasts in the presence of high concentrations of fetal bovine and human whole blood sera. Growth stimulation was observed in plasma-derived serum only when supplemented with exogenous platelet-derived growth factor. Thus, this THP-1 cell product does not fulfill the role of a competence factor.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , División Celular/efectos de los fármacos , Diterpenos , Sustancias de Crecimiento , Leucemia Monocítica Aguda/fisiopatología , Ésteres del Forbol/farmacología , Forboles/farmacología , Terpenos , Neoplasias de la Mama/fisiopatología , Línea Celular , Diploidia , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Humanos , Cinética , Pulmón/embriología , Factor de Crecimiento Derivado de Plaquetas/farmacologíaRESUMEN
Serum-free medium conditioned by activated cells of the acute monocytic leukemia line, THP-1, was examined for growth-inhibitory activity with several established human cell lines. Free-floating clusters of THP-1 cells were activated into adherent nonproliferating cells by a 24-hr exposure to 10(-7) M mezerein in Roswell Park Memorial Institute 1640 medium containing 1% fetal bovine serum. Adherent cells were incubated for an additional 24 hr in serum-free medium containing insulin (5 micrograms/ml). Dose-response studies revealed that a cervical carcinoma (HeLa), a melanoma (A375Ag5), and several mammary carcinoma cell lines (MCF-7, BT474, MDA-MB415, and T47D) were growth inhibited by this conditioned medium. We concluded, from the results of thymidine release assays and from experiments on reversibility, that inhibition was a cytostatic and not a cytolytic response. In contrast, THP-1 conditioned medium stimulated the growth of two mammary lines (ZR75-1 and HBL-100), a lung type II carcinoma (549), and a colon adenocarcinoma (SW48). Preliminary characterization showed that the inhibitory activity was stable to acid and urea treatment but was destroyed by trypsin and sodium dodecyl sulfate. Molecular sieve chromatography of acetic acid-extracted material separated the inhibitory and stimulatory components.