Soluble tumor necrosis factor receptor type I enhances tumor development and persistence in vivo

Secretion of human soluble tumor necrosis factor receptor type I (sTNFRI) by the mouse fibrosarcoma cell line, L929, previously has been demonstrated to confer resistance to in vitro lysis by TNF and to LAK- and CTL-mediated cytolysis. These findings suggest that, in vivo, sTNFRI contributes to tumor survival by inhibiting these immunologic mechanisms. To evaluate this hypothesis, we compared the growth of sTNFRI-secreting L929 cells with that of the unmodified parental fibrosarcoma in an in vivo mouse transplantation model. Secretion of sTNFRI by L929 cells markedly enhanced their tumorigenicity and persistence in syngeneic recipients. This benefit was abrogated by sTNFRI-neutralizing antibodies induced by immunization prior to tumor challenge. These data demonstrate that sTNFRI directly influences tumor formation and persistence in vivo and suggest the selective removal and/or inactivation of sTNFRI as a promising new avenue for cancer immunotherapy.     

Complete amino acid sequence of the human progesterone receptor deduced from cloned cDNA

A lambda gt10 library containing DNAs complementary to messenger RNAs from human breast cancer T47-D cells was constructed and screened with a cDNA probe encoding the rabbit progesterone receptor. Four overlapping clones have been sequenced. The open reading frame corresponds to a protein of 933 amino acids with a molecular weight of 98,868 Da. The cysteine rich basic region supposed to be involved in DNA binding is completely homologous in the human and rabbit receptors, whereas the C-terminal end, where hormone binding is thought to take place, differs by a single amino acid change. The human progesterone receptor is characterized, as is the rabbit receptor, by the very high proline content of its N-terminal region. When mRNAs from either human breast cancer cell lines T47-D and MCF-7 or from normal human uterus tissue were blotted and probed with the cloned cDNA, four main bands were observed (5100, 4300, 3700, and 2900 nucleotides).     

Complete amino acid sequence of the type III isozyme of rat hexokinase, deduced from the cloned cDNA

Clones containing cDNA coding for the Type III isozyme of rat hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) were isolated from a library prepared in lambda gt10 with rat liver mRNA. Three clones were characterized. Their composite sequence includes the entire coding region for Type III hexokinase, 3' untranslated sequence extending into the polyadenylated region, and 80 bp of 5' untranslated sequence. Extensive similarity in sequence of N- and C-terminal halves of the enzyme, previously seen with the Type I isozyme, is consistent with the view that these 100-kDa mammalian hexokinases are the evolutionary result of duplication and fusion of a gene coding for an ancestral hexokinase having a molecular weight of approximately 50 kDa. Extensive similarities are seen between sequences of the Type I and III isozymes, and those reported for mammalian glucokinase (also called Type IV hexokinase) and for the hexokinase and glucokinase of yeast. Residues thought to be involved in catalytic function are highly conserved in all of these enzymes. Based on a quantitative comparison of sequence similarities, it is concluded that the 50-kDa mammalian glucokinase is more closely related to the 100-kDa mammalian enzymes than it is to the 50-kDa enzymes from yeast. One interpretation of this might be that the mammalian glucokinase arose by resplitting of the gene coding for the 100-kDa mammalian hexokinases.     

Effect of cell cycle on the regulation of the cell surface and secreted forms of type I and type II human tumor necrosis factor receptors

The cell cycle has been shown to regulate the biological effects of human tumor necrosis factor (TNF), but to what extent that regulation is due to the modulation of TNF receptors is not clear. In the present report we investigated the effect of the cell cycle on the expression of surface and soluble TNF receptors in human histiocytic lymphoma U-937. Exposure to hydroxyurea, thymidine, etoposide, bisbensimide, and democolcine lead to accumulation of cells primarily in G₁/S, S, S/G₂/M, G₂/M, and M stages of the cell cycle, respectively. Whilie no significant change in TNF receptors occurred in cells arrested in G₁/S or S/G₂ stages, about a 50% decrease was observed in cells at M phase of the cycle. Scatchard analysis showed a reduction in receptor number rather than affinity. In contrast, cells arrested at S phase (thymidine) showed an 80% increase in receptor number. The decrease in the TNF receptors was not due to changes in cell size or protein synthesis. The increase in receptors, however, correlated with an increase in total protein synthesis (to 3.8-fold of the control levels). A proportional change was observed in the p60 and p80 forms of the TNF receptors. A decrease in the surface receptors in cells arrested in M phase correlated with an increase in the amount of soluble receptors. The cellular response to TNF increased to 8- and 2-fold in cells arrested in G₁ and S phase, respectively; but cells at G2/M phase showed about 6-fold decrease in response. In conclusion, our results demonstrate that the cell cycle plays an important role in regulation of cell-surface and soluble TNF receptors and also in the modulation of cellular response. © 1995 Wiley-Liss, Inc.     

Purification and partial amino acid sequence analysis of two distinct tumor necrosis factor receptors from HL60 cells

Two distinct tumor necrosis factor (TNF) receptors of 55- and 75-kDa apparent molecular masses previously identified on the cell surface by monoclonal antibodies have been solubilized with Triton X-100 from HL60 cells. A filter-based dot blot assay was developed to monitor specific 125I-TNF alpha binding during fractionation of the cell extract. By a combination of immuno- and ligand affinity chromatography and reverse phase high performance liquid chromatography both receptor proteins were purified to apparent homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two bands at 55 and 51 kDa for the 55-kDa TNF receptor and a major 75-kDa and a minor 65-kDa band for the 75-kDa TNF receptor. All these bands specifically bound TNF alpha and TNF beta in ligand blot experiments. The exclusive specificity of monoclonal antibodies of the utr series for the 75.65-kDa bands and of the htr series for the 55.51-kDa bands was demonstrated with the purified antigens on Western blots. Both TNF receptor types were found to contain N-linked carbohydrates. N-terminal amino acid sequence analysis of the 55- and 51-kDa bands of the 55-kDa TNF receptor revealed identical sequences suggesting a possible truncation at the C-terminal end. Two different N-terminal sequences were determined for the 65-kDa band. One corresponded to the published sequence of ubiquitin; the other was therefore assumed to be a unique sequence of the 75-kDa TNF receptor. Additional internal sequences of this receptor were determined after proteolytic cleavage.     

The complete amino acid sequence of the shorter form of human basic fibroblast growth factor receptor deduced from its cDNA

We have isolated a full-length cDNA for human basic fibroblast growth factor (bFGF) receptor-like protein from a human placenta cDNA library. Determination of the nucleotide sequence of the cDNA allows elucidation of the complete amino acid sequence of the receptor (731 amino acids) which has two extracellular immunoglobulin-like domains, a transmembrane domain and an intracellular tyrosine kinase domain. The receptor has remarkable amino acid similarity (98% identity) to the shorter form of murine bFGF receptor reported recently (H.H.Reid et al. (1990) Proc.Natl.Acad.Sci. USA 87, 1596-1600). The receptor described here is expected to be the shorter form of human bFGF receptor.     

Interferon-gamma induces cell surface expression for both types of tumor necrosis factor receptors.

Interferons are known to potentiate various biological effects of tumor necrosis factor (TNF). Recently, two different types of TNF receptors with molecular masses of 60 kDa (p60) and 80 kDa (p80), primarily expressed by epithelial cells and myeloid cells, respectively, have been identified. In the present report, we examined the effect of interferon-gamma (IFN-gamma) on each type of TNF receptor. Our results indicate that IFN-gamma induces TNF receptors on both myeloid (e.g. HL-60) and epithelial cells (e.g. HeLa). Furthermore, by using antibodies specific to each type of receptor, we demonstrate that both TNF receptors are equally inducible by IFN-alpha, IFN-beta and IFN-gamma. Thus, the increase in TNF receptors by interferons may play a role in their synergistic cellular response.     

Complete amino acid sequence of the type II isozyme of rat hexokinase, deduced from the cloned cDNA: comparison with a hexokinase from novikoff ascites tumor

The 917-residue amino acid sequence of the Type II isozyme of rat hexokinase has been deduced from the nucleotide sequence of cloned cDNA. The sequences of 197 nucleotides in the 5' untranslated region and 687 bases of the 3' untranslated region have also been determined. A region of overlap between two discrete cDNA clones was confirmed by isolation and sequencing of a genomic DNA clone that spanned the region. Within this region, the 634-nucleotide coding sequence was divided into three exons, each of 150-250 nucleotides; these results suggest that the gene encoding Type II hexokinase is likely to be quite complex. There is extensive similarity between the sequences of the N- and C-terminal halves of the Type II isozyme, as previously seen with the Type I and III isozymes; this is consistent with the view that these enzymes evolved by a process of gene duplication and fusion. A cDNA encoding the entire C-terminal half of a hexokinase from Novikoff ascites tumor cells was also isolated and found to be identical to a cDNA encoding the corresponding region of the Type II isozyme of skeletal muscle. Northern analysis indicated that a single mRNA, approx 5200 nucleotides in length, encoded both the skeletal muscle and the tumor enzymes. These results do not support previous speculation that the hexokinase isozymes of normal tissue are distinct from those of tumors, and suggest the possibility that post-translational modifications of a single protein species might account for apparent differences between the isozymes of normal and tumor tissues.     

The A20 cDNA induced by tumor necrosis factor alpha encodes a novel type of zinc finger protein

We have previously identified in endothelial cells a gene product designated A20, that is rapidly and profoundly induced by tumor necrosis factor alpha (Dixit, V. M., Green, S., Sarma, V., Holzman, L. B., Wolf, F. W., O'Rourke, K., Ward, P. A., Prochownik, E. V., and Marks, R. M. (1990) J. Biol. Chem. 265, 2973-2978). Isolation of the full-length cDNA followed by sequence analysis has revealed that A20 codes for a novel zinc finger protein. Structural features suggest that this putative protein, which contains multiple Cys2/Cys2 finger motifs, defines a novel class of zinc finger proteins. Southern analysis supports the existence of other members of the class.     

The gene for the type 1 tumor necrosis factor receptor (TNF-R1) is localized on band 12p13

Summary The gene coding for the type I (p55) tumor necrosis factor receptor (TNF-R1) has been localized on human chromosome 12, band 12p13.2, by in situ hybridisation using a biotinylated genomic probe.     

Exercise training increases membrane bound form of tumor necrosis factor-alpha receptors with decreases in the secretion of soluble forms of receptors in rat adipocytes

We examined the effects of exercise training (treadmill running over 9 weeks) on the ability of isolated adipocytes to secrete tumor necrosis factor-alpha (TNF-alpha) and type 1 soluble TNF receptor (sTNFR1) in vitro in Wistar rats. We also examined the effects of exercise training on the expression of membrane bound forms of type 1 TNF receptor (mTNFR1) in adipocyte crude membranes of the same rat subjects. Exercise training significantly increased the secretions of TNF-alpha from isolated adipocytes. Treatment with a cyclooxygenase inhibitor, either indomethacin (100 microM) or eicosatetraynoic acid (100 microM), significantly blocked the release of TNF-alpha from adipocytes in both exercise-trained rat group and sedentary control rat group, suggesting that some cyclooxygenase metabolite(s) acts as a ligand in TNF-alpha synthesis. Decreased amounts of TNF-alpha were found to be significantly greater in both exercise-trained rat group than in sedentary control rat group after incubation with inhibitors. Thus, the inhibitory effect of both indomethacin and eicosatetraynoic acid was significantly greater in adipocytes from exercise-trained rats. Both plasma sTNFR1 levels and adipocytes-derived sTNFR1 were found to be significantly less in the exercise-trained rat group. Western blot analysis revealed that exercise training remarkably increased the expressions of mTNFR1 in adipocyte crude membrane. Thus, exercise training enhanced the ability of isolated adipocytes to secrete TNF-alpha with reduced secretion of sTNFR1, and provoked the greater expressions of mTNFR1 in adipocyte crude membrane. These alterations may induce enhanced the autocrine effects of TNF-alpha within adipocytes in exercise-trained rats.     

Modulation of two forms of tumor necrosis factor receptors and their cellular response by soluble receptors and their monoclonal antibodies.

Recently, two different receptors for human tumor necrosis factor (TNF) with molecular masses of 60 kDa (p60) and 80 kDa (p80) have been identified. In this report, we investigated the effect of the soluble forms of these receptors and monoclonal antibodies against them on ligand interaction, receptor down-regulation, and mediation of cellular response in U-937 cells. Our results indicate that p60 and p80 constitute 20-30 and 60-80% of the total TNF-binding sites on U-937 cells, respectively. However, by cross-linking, only the p80 form of the receptor could be detected. In contrast to unlabeled TNF, the anti-p60 and anti-p80 antibodies together only partially inhibited ligand binding, and this inhibition was not additive. Lack of additive inhibition of binding was found to be not due to stereo-chemical hindrance. TNF binding to cells can be completely displaced by soluble forms of either the p60 or p80 receptor. However, 100-fold more of the p80 than the p60 form of the soluble receptor is needed for equivalent displacement. Under optimum conditions, TNF and the anti-p80 and anti-p60 antibodies down-regulated 30, 80, and 20% of the TNF receptors, respectively. The anti-p60 and anti-p80 antibodies down-regulated not only their own receptors, but also reciprocal receptors, suggesting a cross-communication between the p60 and p80 forms of the TNF receptor. In spite of inhibiting as much as 80% of TNF binding, none of the receptor antibodies significantly inhibited the cytotoxic response to TNF in U-937 cells. Soluble forms of both receptors, however, completely abrogated the cellular response to TNF. Thus, overall, our results indicate that the antibodies against both receptors together inhibit the majority of the receptor-ligand interaction without any significant effect on the biological response to TNF.     

Preoperative plasma levels of soluble tumor necrosis factor receptor type I (sTNF-RI) predicts adverse events in cardiac surgery

OBJECTIVE: The objective was to estimate the sTNF-RI preoperative measure in the identification of patients with bad outcome and death. METHODS: We assessed prospectively sixty-two patients submitted electively to myocardial revascularization with ECC or heart valve surgery. The sTNF-RI levels were determined by the Sandwich-Type ELISA method before anesthetic induction. Clinical, surgical characteristics and sTNF-RI levels were compared among patients with good (group I, n=46) or bad outcome (group II, n=16--length of stay in the ICU for over 72 h or death). RESULTS: No difference was found between the verified mortality (6.4%) and the predicted by EuroSCORE (3.0%), p=0.48. The sTNF-RI levels were higher in group II (1322) than group I (748) p=0.009 (levels >954, 69% sensitivity and 70% specificity for good outcome, 44% positive predicted value and 85% negative). The sTNF-RI levels were higher in patients who died (1556) versus (759) p=0.029, (levels >1230, 79% sensitivity, 75% specificity, 20% positive predicted value and 98% negative). In the multivariate logistic regression model sTNF-RI (OR=1.002, IC95% 1.000-1.005, p=0.014) and age (OR=1.083, IC95% 1.010-1.161, p=0.025) were independently related to the risk of bad outcome. CONCLUSIONS: Basal levels of sTNF-RI yield prognostic information in patients who undergo heart surgery.     

Antibodies to a soluble form of a tumor necrosis factor (TNF) receptor have TNF-like activity.

Immunological cross-reactivity between tumor necrosis factor (TNF) binding proteins which are present in human urine (designated TBPI and TBPII) and two molecular species of the cell surface receptors for TNF is demonstrated. The two TNF receptors are shown to be immunologically distinct, to differ in molecular weight (58,000 and 73,000), and to be expressed differentially in different cells. It is further shown that polyclonal antibodies against one of the TNF binding proteins (TBPI) display, by virtue of their ability to bind the TNF receptor, activities which are very similar to those of TNF. These antibodies are cytotoxic to cells which are sensitive to TNF toxicity, induce resistance to TNF toxicity, enhance the incorporation of thymidine into normal fibroblasts, inhibit the growth of chlamydiae, and induce the synthesis of prostaglandin E2. Monovalent F(ab) fragments of the polyclonal antibodies lack TNF-like activities, but acquire them upon cross-linking with anti-F(ab)2 antibodies, suggesting that the ability of the anti-TBPI antibodies to mimic TNF correlates with their ability to cross-link the TNF receptors. This notion was further supported by data obtained in a comparative study of the TNF-like cytotoxicity of a panel of monoclonal antibodies against TBPI. The induction of TNF-like effects by antibodies to a TNF receptor suggests that TNF is not directly involved in intracellular signalling. Rather, it is the receptors to this cytokine which, when properly triggered in a process which appears to involve clustering of these receptors, transduce the signal for response to TNF into the cell's interior.     

The complete amino acid sequence of the catalytic domain of rat brain hexokinase, deduced from the cloned cDNA

The complete amino acid sequence of the catalytic domain of rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) has been deduced from the nucleotide sequence of cloned cDNA. Extensive similarity in sequence, taken to indicate similarity in secondary and tertiary structure, is seen between the mammalian enzyme and yeast hexokinase isozymes A and B. All residues critical for binding glucose to the yeast enzyme are conserved in brain hexokinase. A location for the substrate ATP binding site is proposed based on relation of structural features in the yeast enzyme to characteristics commonly observed in other nucleotide binding enzymes; sequences in regions proposed to be important for binding of ATP to the yeast enzyme are highly conserved in brain hexokinase.     

Interactions between PEG and type I soluble tumor necrosis factor receptor: modulation by pH and by PEGylation at the N terminus

The effects of polyethylene glycol (PEG) on protein structure and the molecular details that regulate its association to polypeptides are largely unknown. These issues were addressed using type I soluble tumor necrosis factor receptor (sTNF-RI) as a model system. Changes in solution viscosity established that a truncated form of sTNF-RI bound free PEG in a pH-dependent manner. Above pH 5.3, the viscosity escalated as the pH increased, while no effect occurred below pH 5.0. Conjugation of 2 kD, 5 kD, or 20 kD PEG to the N terminus attenuated the viscosity at the higher pH values. Tryptophan phosphorescence spectroscopy correlated changes in the protein structure about Trp-107, at the C terminus, with the pH-dependent and PEGylation-dependent attenuation of the viscosity. The results indicate that specific interactions between PEG and the truncated form of sTNF-RI are elicited by an increased flexibility of the truncated protein combined perhaps with removal of steric or charge barriers. Covalently bound PEG at the N terminus reduced the protein affinity for the free polymer and induced a more rigid and polar configuration around Trp-107. Deprotonation of His-105, which is perpendicular to Trp-107, was integral to the binding mechanism producing a pH-dependent switching mechanism. These findings stress the importance of surface charge and structural plasticity in determining macromolecular binding affinities and demonstrate the ability of conjugated PEG to modify the localized surface structure in proteins away from the site of conjugation.     

Role of amino acid residue 90 in bioactivity and receptor binding capacity of tumor necrosis factor mutants

We have previously produced two bioactive lysine-deficient mutants of TNF-alpha (mutTNF-K90R,-K90P) and found that these mutants have bioactivity superior to wild-type TNF (wtTNF). Because these mutants contained same amino acid except for amino acid 90, it is unclear which amino acid residue is optimal for showing bioactivity. We speculated that this amino acid position was exchangeable, and this amino acid substitution enabled the creation of lysine-deficient mutants with enhanced bioactivity. Therefore, we produced mutTNF-K90R variants (mutTNF-R90X), in which R90 was replaced with other amino acids, to assay their bioactivities and investigated the importance of amino acid position 90. As a result, mutTNF-R90X that replaced R90 with lysine, arginine and proline were bioactive, while other mutants were not bioactive. Moreover, these three mutants showed bioactivity as good as or better than wtTNF. R90 replaced with lysine or arginine had especially superior binding affinities. These results suggest that the amino acid position 90 in TNF-alpha is important for TNF-alpha bioactivity and could be altered to improve its bioactivity to generate a "super-agonist".     

Processing of tumor necrosis factor by the membrane-bound TNF-alpha-converting enzyme, but not its truncated soluble form.

Tumour necrosis factor (TNF)-alpha-converting enzyme (TACE) is a membrane protein belonging to the ADAM (a disintegrin and metalloproteinase) family that cleaves various membrane proteins, including the proform of TNF-alpha. In this study, we constructed expression vectors for the membrane-bound full-length TACE (mTACE) and its truncated soluble form (sTACE). When a human TNF-alpha expression vector was introduced into human 293 cells, processing of TNF-alpha to its mature form was enhanced by coexpressing mTACE, and this processing was inhibited by a metalloproteinase inhibitor. On the other hand, coexpression of sTACE had no effect on the processing of TNF-alpha, although the culture medium of sTACE-transfected cells could cleave a peptide containing the TNF-alpha cleavage site. Fas ligand (FasL)-transfected 293 cells released a considerable amount of soluble FasL, and coexpression of neither mTACE nor sTACE enhanced this shedding. Immunoprecipitation and Western blotting analysis with cells that were cotransfected with TACE and TNF-alpha indicated that both mTACE and sTACE could interact with the proform of TNF-alpha. In the same assay, neither mTACE nor sTACE interacted with FasL. The catalytic domain-lacking TACE mutant, which could also interact TNF-alpha, showed a dominant negative effect on not only TNF-alpha secretion but also FasL secretion. These results suggest that binding of the membrane-anchored but not the soluble form of TACE to TNF-alpha results in efficient ectodomain shedding, and that FasL secretase is a metalloproteinase similar, but not identical, to TACE.     

Alterations of events related to ovarian function in tumor necrosis factor receptor type I knockout mice

C57BL6 mice with targeted disruption of tumor necrosis factor (TNF) type 1 receptor (TNFRI) exhibited early vaginal opening when compared with wild-type mice (Day 24 +/- 0.6, n = 10, vs. 28 +/- 0.2, n = 11, P < 0.001). Equine CG- and hCG-treated TNFRI null mice ovulated more ova than did controls at two distinct times during the prepubertal period (Day 21: 13.4 +/- 1.7 vs. 7.3 +/- 1.4, P < 0.05; Day 25: 20.7 +/- 2.7 vs. 13.0 +/- 1.3, P < 0.05). Enhanced responsiveness to gonadotropins was not observed in adult mice. At 6 mo of age only 40% of TNFRI null mice exhibited estrous cycles. Those TNFRI null mice with estrous cycles spent significantly more time in diestrus and less time in estrus than controls. TNFRI null mice delivered significantly fewer litters (P < 0.001) than did C57BL6 and TNFRII null mice (TNFRI null 2.59 +/- 0.39; C57BL6 4.91 +/- 0.57; TNFRII null 5.40 +/- 0.60 litters/mo/10 pairs over a 12-mo period). Ovarian dispersates prepared on Day 25 of age from control and TNFRI knockout mice were cultured with and without 10 ng TNF/ml. TNF inhibited LH-stimulated progesterone and estradiol secretion by control dispersates but had no effect on cAMP. In contrast, TNF did not affect LH-stimulated accumulation of progesterone, estradiol, or cAMP by ovarian dispersates from TNFRI knockout mice. The results indicate that lack of TNFRI enhances ovarian responsiveness to gonadotropins during the prepubertal period and may be related to early vaginal opening. The lack of TNFRI is associated with early senescence and poor fertility. These studies demonstrate that the mechanism of TNF-mediated inhibition of steroidogenesis is most likely via TNFRI.     

The amino acid sequence of nucleoside diphosphate kinase I from spinach leaves, as deduced from the cDNA sequence.

The primary structure of nucleoside diphosphate (NDP) kinase from spinach leaves has been deduced from its cDNA sequence. A lambda gt 11 cDNA library derived from spinach leaves was screened using an antibody against NDP kinase I, which we previously purified to electrophoretic homogeneity (T. Nomura, T. Fukui, and A. Ichikawa, 1991, Biochim. Biophys. Acta 1077, 47-55). The cDNA sequences of positive clones contained the amino acid coding region (444 base pairs) for NDP kinase I as well as 5' and 3' noncoding regions of 33 and 361 base pairs, respectively. The cDNAs hybridized to a 1.1-kb mRNA. NDP kinase I contains 148 amino acid residues with a molecular mass of 16,305, which is in excellent agreement with that of the purified enzyme (16 kDa). Homology was found between the sequence of spinach NDP kinase I and those of the rat, Myxococcus xanthus, and Dictyostelium discoideum NDP kinases, as well as the human Nm23-gene product and the awd protein of Drosophila melanogaster.