Analysis of the structure-function relationship of tumour necrosis factor. Human/mouse chimeric TNF proteins: General properties and epitope analysis (original) (raw)
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Tumor necrosis factor Characterization at the molecular, cellular and in vivo level
Febs Letters, 1991
TNF was originally characterized as an antitumor agent and a factor cytotoxic for many malignant cells. It is now clear that it plays an important role in the defense against viral, bacterial and parasitic infections, -and in (auto-)immune responses. Natural induction of TWF is protective, but its overproduction may he detrimental and even lethal to the host. The steucture of TNF and its interaction with the two types of cellular receptor are becoming better understood. TNF elicits a variety of events in different cell types. It subverts the electron transport system or the mitochondria into production of oxygen radicals, which can kill the (malignant) cells when these do not contain or produce protective enzymes. Furthermore, TNF induces a set of genes and at least part of this transcriptional activation is mediated by NFKB. The prospects of TNF as an antitumor drug can be improved on the one hand by agents such as LI+, which synergizes, and on the other hand by inhibitors of the systemic toxicity which do not interfere with the antitumor efficacy. Also, in tumor-bearing animals which have been rendered tolerant by administration of small doses of TNF, an effective and complete elimination of the tumors can be obtained by the combined action of TNF plus interferon.
Molecular cloning of mouse tumour necrosis factor cDNA and its eukaryotic expression
Nucleic Acids Research, 1985
Tumour necrosis factor (TNF), released by induced macrophages, causes tumour necrosis in animals and kills preferentially transformed cells in vitro. mRNA induced in the established mouse monocytic PU 5.1.8 cell line by lipopolysaccharide, was converted into double-stranded cDNA and cloned in the pAT153 vector. Recombinant plasmids were screened by plus-minus hybridization and TNF-specific oligonucleotide probes constructed on the basis of partial amino acid sequences of rabbit TNF. A series of TNF specific clones were identified and confirmed by hybrid selection of mouse TNF-specific mRNA. The sequence codes for a 235 amino acids long polypeptide, of which 156 amino acids presumably correspond to the mature product. It can be concluded that mature mouse TNF is a glycosylated dimer. Biologically active TNF was secreted by both Cos-I and CHO-cells transfected with the chimaeric expression vector pSV2d2-mTNF containing the coding region of the mouse TNF cDNA gene.
In vitro anti-tumour activity of tumour necrosis serum
International Journal of Immunopharmacology, 1980
A method measuring 3H-thymidine incorporation in Meth A sarcoma cells was used to quantify in vitro anti-tumour activity of tumour necrosis serum and compared with a method using cell viability as a parameter. Tumour necrosis serum obtained from mice pretreated with Corynebacterium parvum and elicited with endotoxin two weeks later greatly inhibited 3H-thymidine incorporation, whereas sera of normal mice and of mice treated with C. parvum or endotoxin alone were much less inhibitory. All sera reduced viable cell numbers, tumour necrosis serum being most active. The 3H-thymidine incorporation assay is suited for screening mouse sera on anti-tumour activity. It was shown that the anti-tumour activity of normal mouse serum can be potentiated by in vivo pretreatment of mice with bacterial agents. The mechanism of the anti-tumour action(s) and the factor(s) involved remain to be elucidated.
FEBS Letters, 1996
In order to map the immunogenic epitope for the produced mainly by activated monocytes-macrophages, was monoclonal antibody E7H2 on the human turnout necrosis factor initially characterized as a protein inducing the haemorrhagic (hTNF-~) molecule, a number of chimeric proteins were necrosis of certain transplanted tumours in mice [7]. To date, developed by in-frame joining segments of the human genes TNF-c~ has been proved to be a key mediator of inflammation encoding TNF-c~ and lymphotoxin (TNF-[3) as well as by during infectious diseases and exerts strong system effects incoupling appropriate coding regions for human and mouse cluding the septic shock [8]. In an attempt to gain more in-TNF-c~. High level expression of these chimeric genes was sight into the functional topography of the TNF-~ molecule, achieved in Escherichia coli by placing the coding sequences we initiated an epitope analysis using monoclonal antibodies under control of either E. coli trp-promoter or a tandem of (Mabs) E7H2 [9] which are specific for human recombinant bacteriophage T7 constitutive promoters A2 and A3. As revealed TNF-~ [10]. Here, we report the construction of chimeric and by Western blot analysis with monoclonal antibody E7H2 mutant TNF molecules and their use for defining the epitope directed against human TNF-~, the region involved in the binding of this antibody includes sequence ValGluLeuArg in the recognized by Mabs E7H2. N-terminal part of the TNF-ot molecule. 2. Materials and methods
Oligomeric tumer necrosis factor α slowly converts into inactive forms at bioactive levels
Biochemical Journal
The stability of oligomeric human tumour necrasis factor a (TNF) at bioactive levels has been studied by two immunoenzymatic assays: one able to specifically detect oligomeric and not monomeric TNF (O-e.l.i.s.a.) and the other able to detect both forms (OM-e.l.i.s.a.). The selectivity of O-e.l.i.s.a. and OM-e.l.i.s.a. for oligomeric and monomeric TNF was demonstrated with isolated forms prepared by partial dissociation of recombinant TNF with 10% (v/v) dimethyl sulphoxide and gel-filtration h.p.l.c. Evidence for instability of oligomeric TNF were obtained in physiological buffers, as well as in serum and cell-culture supernatants, as a function of TNF concentration. In particular, only a half of the TNF antigen was recovered in the oligomeric form after 72 h incubation (37°C) at 0.12 nm, whereas no apparent dissociation was detected at 4 nm. The structural changes observed at picomolar concentrations were rapidly reversed by raising the concentration of TNF to about 2 nm by ultrafiltration, suggesting that subunit dissociation and reassociation reactions occur in the picomolar and nanomolar range respectively. The cytolytic activity of L-M cells correlates with oligomeric-TNF levels after incubation at picomolar concentrations. Moreover, isolated oligomeric TNF was cytotoxic towards L-M cells, whereas monomeric TNF was virtually inactive. In conclusion, the results suggest that bioactive oligomeric TNF is unstable at picomolar levels and slowly converts into inactive monomers, supporting the hypothesis that quaternary-structure changes in TNF may contribute to the fine regulation of TNF cytotoxicity.
Purification and characterization of a human tumor necrosis factor from the LuKII cell line
Proceedings of the National Academy of Sciences, 1985
A factor with tumor necrosis factor (TNF) activity produced by the LuKII human lymphoblastoid cell line [designated TNF(LuKII)] was purified sequentially by using controlled-pore glass, lentil lectin-Sepharose, and procion red agarose chromatography, yielding TNF with a specific activity of 1.5 X 10(7) units per mg of protein and an isoelectric point of approximately equal to 6.7. Purified TNF(LuKII) fractionated by NaDodSO4/PAGE under reducing as well as nonreducing conditions was found to contain seven protein bands of Mr 80,000, 70,000, 43,000, 25,000, 23,000, 21,000, and 19,000. The proteins of Mr 80,000 and 70,000 could not be dissociated into lower molecular weight components. Peptide mapping analysis and immunoblotting analysis revealed that the seven protein bands in the purified TNF(LuKII) preparations are related. After fractionation of TNF(LuKII) by NaDodSO4/PAGE under reducing conditions, TNF activity was recovered from the regions of Mr 70,000 and 19,000-25,000. Purifie...