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Biomedical subjects

W Fiers

Publications and source records attributed to W Fiers.

At least 307 records · Page 17Linked to original sources
PubMed1986

Induction of the synthesis of tumor necrosis factor receptors by interferon-gamma.

Human HT-29 colon carcinoma and HeLa D98/AH2 and SK-MEL-109 melanoma cells were sensitive to synergistic growth inhibition by concentrations of recombinant human tumor necrosis factor (rTNF) and interferon-gamma (rIFN-gamma) which individually were only slightly inhibitory. We investigated whether this synergism could be explained by the presence of an increased number of TNF receptors in cells treated with rIFN-gamma. These receptors were measured by incubating cells resuspended from monolayers with 125I-rTNF. HT-29 cells treated for a few hours with rIFN-gamma could bind more 125I-rTNF than control untreated cells, but this binding returned to the level of control cells after 24 hr. The treatment with rIFN-gamma did not change the binding affinity of TNF receptors, but increased their number to 1800 per cell from a basal level of about 800 per cell. Inhibitors of RNA synthesis prevented this increase. HT-29 cells were significantly more growth-inhibited when treated first for 6 to 12 hr with rIFN-gamma and then with rTNF, than when treated first with rTNF and then with rIFN-gamma. Untreated HeLa D98/AH2 and SK-MEL-109 cells had 2400 and 9000 receptors per cell, with a KD similar to that of HT-29 cells (approximately 2 X 10(-10)M). A significant increase in TNF receptors after treatment with rIFN-gamma was observed in HeLa D98/AH2, but not in SK-MEL-109 cells. No increase in TNF receptors was detected in cells treated with rIFN-alpha 2. These results indicate that the synergism between rTNF and rIFN-gamma may be due, at least in part, to a transient induction of the synthesis of TNF receptors by rIFN-gamma in cells with a relatively low number of these receptors.

Adenocarcinoma↗
PubMed1986

Inefficient translation initiation causes premature transcription termination in the lacZ gene.

Expression plasmids containing the E. coli lacZ coding region preceded by a set of different ribosome-binding sites and put under transcriptional control of the leftward promoter of phage lambda (PL) were used to study the synthesis of lacZ mRNA. In a normal host the steady state level of full-length lacZ mRNA varied 100-fold with the different synthesis levels of beta-galactosidase, whereas in a host expressing the antitermination protein N of phage lambda, all vectors synthesized the same amount of full-length lacZ mRNA, while maintaining the differences in beta-galactosidase expression. We present evidence for a causal relationship between the rate of ribosome loading and the continuation of transcription across the lacZ gene. We suggest that extended spacing between the RNA polymerase and the elongating ribosome causes transcriptional polarity by increasing the extent of premature termination. The conditional character of the termination event can best be explained by invoking termination factor Rho.

Galactosidases↗
PubMed1986

Synthesis and maturation of recombinant human tumor necrosis factor in eukaryotic systems.

The biosynthesis of human tumor necrosis factor (hTNF) was studied. The amino-terminal extension of the hTNF precursor (26 kDa polypeptide) was not cleaved off in a cell-free system supplemented with dog pancreas microsomes. Correct maturation of pre-hTNF was nevertheless not restricted to the macrophage system: in the medium of a TNF-producing, transformed CHO cell line, a (weak) approximately 20 kDa, an approximately 18.5 kDa (doublet) and a 17 kDa TNF polypeptide, the latter corresponding to mature hTNF, were revealed by specific immunoprecipitation. Similar results were obtained with Xenopus laevis oocytes, injected with hTNF mRNA, except that the 20 kDa band was lacking. The results are discussed in relation to the secretion mechanism of hTNF.

Animals↗
PubMed1986

Two distinct monokines, interleukin 1 and tumor necrosis factor, each independently induce biosynthesis and transient expression of the same antigen on the surface of cultured human vascular endothelial cells.

A murine monoclonal antibody (H4/18) raised against cultured human endothelial cells (HEC) prestimulated by the monokine interleukin 1 (IL 1) recognizes a cell surface molecule inducible by IL 1 or by the distinct monokine tumor necrosis factor (TNF) in primary or serially passaged HEC. H4/18 binding is not basally expressed or inducible by IL 1 in an SV-40 transformed HEC line, in human dermal fibroblasts, or in blood leukocytes. Expression of this molecule by HEC in response to IL 1 can be blocked by protein and RNA synthesis inhibitors but not by cyclooxygenase inhibitors. In addition, H4/18 can immunoprecipitate two biosynthetically labeled polypeptides (Mr 100,000 and 120,000) from HEC stimulated with IL 1 but not from control HEC. Thus, the H4/18 binding site appears to be an inducible surface protein specific for HEC. The majority of HEC in a culture can be induced to express the H4/18 binding protein, but expression is transient (peak 4 to 6 hr) and over the next 24 hr declines to near basal levels either in the continued presence of or upon removal of IL 1. The magnitude of the peak response depends upon IL 1 concentration (peak 5 to 10 U/ml), and the response is optimized by the continued presence of IL 1 during the initial 4- to 6-hr induction period. The time of peak H4/18 binding does not appear to be a function of IL 1 concentration. The decline of H4/18 binding from peak levels is prevented by cycloheximide, a protein synthesis inhibitor. HEC maintained in the presence of IL 1 for 24 hr become refractory to restimulation by IL 1; however, IL 1-stimulated cells rested in the absence of IL 1 for 20 hr can be stimulated by fresh IL 1. HEC expression of the H4/18 binding protein is not induced by interleukin 2 or by interferon-alpha, -beta, or -gamma. Induction of H4/18 binding by TNF is also concentration dependent, transient, and dependent upon protein and RNA synthesis. Several observations suggest that IL1 and TNF act independently on HEC. Our TNF is a recombinant protein, expressed from a cloned cDNA and thus free of IL 1 contamination; it also has no activity in a highly sensitive IL 1 assay. Our standard IL 1 preparation is affinity purified and lacks TNF activity on L929 cells. Thus, our monokine preparations are not cross-contaminated. Most interestingly, HEC incubated with IL 1 and refractory to IL1 restimulation can be restimulated by TNF to express H4/18 binding and vice versa.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Cloning and structure of a mouse interleukin-2 chromosomal gene.

Using non-stringent hybridization with a human interleukin-2 cDNA probe, we have isolated recombinant phages from a mouse genomic DNA library cloned in the EMBL3 phage. The sequence and organization of the mouse interleukin-2 (IL-2) gene was determined. By comparison with the human IL-2 sequence, three introns can be identified with lengths of 99, +/- 2 400, and +/- 1 900 base pairs, respectively. The mouse IL-2 gene codes for a polypeptide of 169 amino acids and contains a putative signal peptide of 20 amino acids. The homology to the human interleukin-2 is 72% at the nucleotide level in the coding part and 65% at the amino acid level. An extraordinary sequence, consisting of 12 consecutive CAG codons coding for glutamine, is found in the first exon.

Amino Acid Sequence↗
PubMed1986

Recombinant tumor necrosis factor: species specificity for a variety of human and murine transformed cell lines.

Tumor necrosis factor (TNF) exhibits cytotoxic or cytostatic activity on a wide range of animal and human transformed cell lines. Using pure, recombinant human and mouse TNF, we examined the degree of species specificity of the in vitro TNF activity on a variety of human and murine transformed cell lines. This species specificity was studied for the TNF activity alone or in synergism with IFN-gamma. Recombinant human and mouse TNF behave remarkably similarly regarding the in vitro cytolytic/cytostatic activity. However, a certain degree of species-specific preference could be revealed as human cell lines needed a higher concentration of recombinant mouse TNF than of recombinant human TNF to attain a similar effect, while on mouse cells the reverse was true. Also, synergism with IFN-gamma seemed more effective when the target cell was treated with homologous TNF.

Animals↗
PubMed1986

Natural killer cell response to interferons.

The availability of recombinant interferons has facilitated a comparison of the in vitro effects of interferon-alpha and -gamma upon human natural killer (NK) cells. In the absence of interferon high and low NK responders exist. Repeated testing of the same individuals revealed different but stable NK patterns to in vitro addition of interferon-alpha and -gamma Furthermore, the NK cell response patterns differed depending on whether the interferons were administered in combination or separately. These studies suggest new complexities that must be addressed in the planning and execution of clinical interferon trials. The pattern of responsiveness of human NK cells to interferon-alpha or -gamma or both is highly variable, but stable, within a given individual.

Cytotoxicity, Immunologic↗
PubMed1986

Recombinant tumor necrosis factor: its effect and its synergism with interferon-gamma on a variety of normal and transformed human cell lines.

Tumor Necrosis Factor (TNF), released by induced macrophages, causes tumor necrosis in animals, and preferentially kills transformed cells in vitro. Using pure, recombinant human TNF, we report here its cytotoxic action on several human transformed and non-transformed cell lines. Furthermore, remarkable synergism between TNF and interferon-gamma (IFN-gamma) was observed in a large number of human cell lines, especially breast, cervix and colon carcinomas. Some other human cell lines, not sensitive to TNF alone, became highly sensitive when IFN-gamma was present as well. We could not demonstrate a synergism between TNF and IFN-gamma on any of the lymphoma/leukemia cell lines tested. All normal human, non-transformed diploid cell lines were insensitive to TNF even in the presence of IFN-gamma. This study also confirms the observation that inhibition of protein synthesis by metabolic drugs (e.g. actinomycin D) remarkably enhances the sensitivity of several target cell lines to cytolysis by TNF.

Cell Division↗
PubMed1986

Construction of stable laboratory and industrial yeast strains expressing a foreign gene by integrative transformation using a dominant selection system.

An expression cassette of mouse dihydrofolate reductase (Mdhfr) cDNA under control of the yeast cytochrome c promoter was inserted in a yeast plasmid containing the ARS1 sequence. The ARS replicating function was destroyed by BglII treatment prior to yeast transformation. Using this linearized plasmid, genomic transformants could be obtained from either laboratory or industrial strains of bakers' yeast based on direct methotrexate (MTX)-resistance selection. The entire sequence of the linearized plasmid was integrated by homologous recombination at the ARS region of the host chromosome. The results indicate that repetitive and homologous recombination occurs readily in such transformations. The stability of the constructed integrants was more than 99.95% per generation in non-selective medium, and tandem repeats of up to six copies (i.e., about 44 kb) were not changed even after 30 generations in rich medium. Expression in rich medium of cointegrated, human interleukin 2 cDNA under control of the triose phosphate isomerase promoter was shown by Western blot experiments in both laboratory and industrial yeast strains. Furthermore, a comparison of the transcription efficiency of the Mdhfr gene in the chromosome with that in the plasmid revealed that the efficiency was almost proportional to the number of gene copies, irrespective of the location of the transcription unit. These results show that by using the MTX/Mdhfr dominant selection-amplification system one can construct stable recombinant yeast strains suitable for heterologous gene expression in laboratory as well as in industrial fermentation conditions.

DNA, Fungal↗
PubMed1986

Recombinant tumor necrosis factor induces procoagulant activity in cultured human vascular endothelium: characterization and comparison with the actions of interleukin 1.

Human recombinant tumor necrosis factor (rTNF) was found to act directly on cultured human vascular endothelium to induce a tissue factor-like procoagulant activity (PCA). After a 4-hr incubation in rTNF (100 units/ml), serially passaged endothelial cells isolated from umbilical veins, saphenous veins, iliac arteries, and thoracic aortae demonstrated a dramatic increase (4- to 15-fold, 21 experiments) in total cellular PCA as measured with a one-stage clotting assay. rTNF-induced PCA was also expressed at the surface of intact viable endothelial monolayers. Induction of PCA by rTNF was concentration dependent (maximum, 500 units/ml), time dependent, reversible, and blocked by cycloheximide and actinomycin D, and it occurred without detectable endothelial cell damage. Actions of rTNF were compared with those of natural human interleukin 1 (IL-1) derived from stimulated monocytes and two distinct species of recombinant IL-1, each of which also induced endothelial PCA. The use of recombinant polypeptides and specific neutralizing antisera established the distinct natures of the mediators. The kinetics of the endothelial PCA responses to TNF and IL-1 were similar, demonstrating a rapid rise to peak activity at approximately equal to 4 hr, and a decline toward basal levels by 24 hr. This characteristic decline in PCA after prolonged incubation with TNF or IL-1 was accompanied by selective endothelial hyporesponsiveness to the initially stimulating monokine. Interestingly, the effects of TNF and IL-1 were found to be additive even at apparent maximal doses of the individual monokines. Endothelial-directed actions of TNF, alone or in combination with other monokines, may be important in the initiation of coagulation and inflammatory responses in vivo.

Blood Coagulation Factors↗
PubMed1986

Recombinant human tumor necrosis factor increases mRNA levels and surface expression of HLA-A,B antigens in vascular endothelial cells and dermal fibroblasts in vitro.

Recombinant human tumor necrosis factor (TNF), purified to greater than 99% homogeneity, increases surface expression of class I major histocompatibility complex (MHC) antigens to a maximum of 9-fold on cultured human endothelial cells (HEC) and human dermal fibroblasts (HDF). The increase is concentration dependent (peak 20-100 units/ml) and time dependent (nearly maximal by 4 days); expression remains elevated in the continued presence of TNF and requires greater than 7 days to return to basal levels upon TNF withdrawal. The increase in surface expression appears to result from increases in steady-state mRNA levels for the class I antigens, although the increase in mRNA is proportionately greater than for surface expression. No surface expression of or mRNA for class II MHC antigens is detectable in either control or TNF-treated HEC or HDF. These effects are similar to those produced by leukocyte or fibroblast (type I) interferons (IFNs). The protein synthesis inhibitor cycloheximide (CHX), when added coincidentally with type I IFNs, leads to superinduction of mRNA for class I MHC antigens and, unexpectedly, leads to the appearance of mRNA for class II MHC antigens. CHX has no effect by itself upon mRNA levels for class I or class II MHC antigens, nor does it modulate the increases in mRNA produced by immune (type II) IFN. Most interesting, CHX blocks the increase in mRNA for class I MHC antigens induced by TNF. Thus TNF appears to act on MHC gene expression through a newly synthesized protein intermediate. Our results provide direct evidence that TNF can modulate gene expression in normal (untransformed) cell types and contribute to understanding the complex nature of MHC gene regulation. Finally, they suggest that TNF may act in vivo as an immunoregulatory molecule.

Cell Membrane↗
PubMed1986

Genes for the tumor necrosis factors alpha and beta are linked to the human major histocompatibility complex.

The human major histocompatibility complex (MHC) includes the closely linked genes for the tumor necrosis factors alpha and beta. Their location is within the chromosomal segment between HLA-DR and HLA-A or centromeric of HLA-DP. This assignment is based on Southern blot analysis of a number of different MHC deletion mutants and is corroborated by chromosome in situ hybridization.

Chromosome Deletion↗
PubMed1986

Treatment of murine interferon-alpha/beta-sensitive and -resistant friend leukemia cells with tumor necrosis factor in combination with murine interferon-alpha/beta or -gamma.

Interferon-alpha/beta (IFN-alpha/beta)-sensitive (FLC 745) and -resistant (FLC 3Cl8) Friend leukemia cells (FLC) were fairly refractory to the cytotoxicity of murine tumor necrosis factor (MuTNF) in vitro. Both lines became highly sensitive to mTNF when treated in combination with murine IFN-gamma; when treated with IFN-alpha/beta, only the FLC 745 became sensitive to MuTNF. Romeo et al. have reported previously that an antiviral state was induced in both FLC lines by IFN-gamma, but only in FLC 745 by IFN-alpha/beta. The present results suggest that the sensitivity to the synergistic cytotoxic effect of IFN and TNF in these cells is correlated with the ability to be induced into an antiviral state.

Animals↗
PubMed1986

Recombinant tumor necrosis factor and immune interferon act singly and in combination to reorganize human vascular endothelial cell monolayers.

Human endothelial cells (HECs) in confluent primary culture reproduce the epithelioid organization of the vascular lining in situ. The addition of greater than or equal to 20 U/ml recombinant tumor necrosis factor (rTNF) or greater than or equal to 16 U/ml recombinant immune interferon (rIFN-gamma) causes HECs 1) to become elongated, 2) to overlap, 3) to rearrange their actin filaments, and 4) to lose their stainable fibronectin matrix. These changes develop over 72-96 hours and are reversible upon withdrawal of the mediator. In serially passaged HECs similar morphologic changes develop. Furthermore, rTNF and rIFN-gamma are each cytostatic for subconfluent passaged cultures. When added in combination, low concentrations of rTNF and rIFN-gamma act synergistically, whereas higher concentrations (eg, 100 U/ml rTNF and 200 U/ml rIFN-gamma) produce unique morphologic changes. Doubly treated primary HECs extend many long, overlapping, spinelike processes and expose the substratum. Doubly treated passaged HECs become extremely elongated and then shed in large numbers. These in vitro changes in endothelial cell morphology and behavior may underlie immunologically mediated vascular responses in vivo.

Actins↗
PubMed1986

Human tumor xenografts treated with recombinant human tumor necrosis factor alone or in combination with interferons.

We have studied the activity of recombinant human tumor necrosis factor (rHuTNF) on six different human tumor xenografts derived from primary breast and bowel tumors and maintained by passage in nude mice. When 5 micrograms rHuTNF was given daily intratumorally to mice with established (approximately, 0.5 cm) tumors, total tumor regression was observed by 3-4 weeks in three of six xenograft lines. In a further two lines tumor stasis or significant slowing of growth was seen. This antitumor action was not accompanied by any consistent macroscopic change in the tumor such as necrosis, but histological examination revealed tumor cell degeneration and a large peritumoral infiltration of host inflammatory cells after 4-7 days therapy. In contrast to these data, little effect was seen when the same dose of rHuTNF was administered i.p. to nude mice bearing these tumors. In only two of six lines was any significant slowing of tumor growth seen. A 5-fold increase in the i.p. dose resulted in improved activity on only one of two xenograft lines tested. Efficacy of the i.p. rHuTNF dose could, however, be enhanced by simultaneous administration of human interferon, alpha or gamma. No obvious signs of toxicity were observed at all rHuTNF doses administered and weights of control and treated mice at the end of the experiments were comparable.

Animals↗
PubMed1985

Binding of human tumor necrosis factor to high affinity receptors on HeLa and lymphoblastoid cells sensitive to growth inhibition.

Purified human tumor necrosis factor (TNF) was iodinated to high specific activity with good retention of its biological activity, as determined by the cytotoxic titer on murine L929 cells. The binding of 125I-TNF to L929 and human HeLa S2 cells grown in monolayer was initially measured, but high levels of nonspecific binding were observed. Specific binding to high affinity receptors of HeLa S2 cells grown in suspension culture was demonstrated by competitive displacement experiments and analysis of the binding data with the LIGAND program. A KD of 2 X 10(-10) M and 6000 receptors/cell were calculated in this way. These observations provide the first direct evidence for a cellular receptor for TNF. The cell-bound 125I-TNF was internalized at 37 degrees C, presumably by receptor-mediated endocytosis, and subsequently degraded to acid-soluble products. Three lines of human lymphoblastoid cells were examined for sensitivity to the cytostatic effect of TNF and for the presence of high affinity receptors. Daudi and Raji cells were insensitive to TNF and showed very few specific binding sites when incubated with 125I-TNF. Jurkat cells were growth-inhibited by TNF and showed a significantly greater number of specific binding sites than the other lymphoblastoid cells. These findings suggest that the sensitivity of some cell lines to the biological effects of TNF may be correlated with the presence of a relatively high number of receptors for this factor.

Animals↗