Tumor necrosis factor (TNF) is a soluble protein that causes damage to tumor cells but has no effect on normal cells. Human TNF was purified to apparent homogeneity as a 17.3-kilodalton protein from HL-60 leukemia cells and showed cytotoxic and cytostatic activities against various human tumor cell lines. The amino acid sequence was determined for the amino terminal end of the purified protein, and oligodeoxyribonucleotide probes were synthesized on the basis of this sequence. Complementary DNA (cDNA) encoding human TNF was cloned from induced HL-60 messenger RNA and was confirmed by hybrid-selection assay, direct expression in COS-7 cells, and nucleotide sequence analysis. The human TNF cDNA is 1585 base pairs in length and encodes a protein of 233 amino acids. The mature protein begins at residue 77, leaving a long leader sequence of 76 amino acids. Expression of high levels of human TNF in Escherichia coli was accomplished under control of the bacteriophage lambda PL promoter and gene N ribosome binding site.

Human fibroblast interferon has three cysteine residues, located at amino acid positions 17, 31, and 141. Using the technique of site-specific mutagenesis with a synthetic oligonucleotide primer, we changed the codon for cysteine-17 to a codon for serine. The resulting interferon, IFN-fi Ser-17, retains the antiviral, natural killer cell activation, and antiproliferative activities of native fibroblast interferon. The purified IFN-j3 Ser-17 protein has an antiviral specific activity of 2 x 108 units/mg, similar to that of purified native fibroblast interferon. In addition, the purified protein is stable to long-term storage at -700C.In this report we describe the site-specific mutagenesis of the cysteine residue to a serine residue at position 17 in the amino acid sequence of IFN-/3 (Fig. lA). The site-specific mutagenesis is induced by using the synthetic 17-nucleotide primer G-C-A-A-T-T-T-T-C-A-G-A-G-T-C-A-G, which is complementary to 16 of the 17 nucleotides on the sense strand of the IFN-p gene and includes the complementary base triplet of the TGT codon 17 for cysteine. There is a onebase change at nucleotide 12 in the primer from a T to an A to induce the mutation.The human fibroblast interferon (IFN-j3) gene has been cloned and expressed to high levels in Escherichia coli (1-5). However, when the cloned IFN-,3 protein was purified from extracts of E. coli, we found that its antiviral specific activity was only 3 x 107 units/mg, about 1/10th that of the native glycosylated protein (6). In addition, we found most of the IFN-,3 protein existed as dimers and oligomers in E. coli.IFN-P has three cysteine residues, located at amino acid positions 17, 31, and 141 (2). One or more of these cysteines could be involved in intermolecular disulfide bridging, resulting in the formation of inactive dimers and oligomers. In addition, the three cysteines may interact randomly within each molecule, resulting in three types of molecules in the cell, each with one of the three possible intramolecular disulfide bridges. Only one of these forms may resemble the native conformation and retain biological activity. Both of these possibilities could together result in the formation of inactive monomers and oligomers within the cell.To investigate if the sulfhydryls are responsible for the lower specific activity of the IFN-,B protein by these mechanisms, we sought to remove one of the three cysteine residues by site-specific mutagenesis of the IFN-p gene, changing one of the codons for cysteine to that for seine. The resulting interferon protein has only two cysteine residues, and therefore can form only a unique intramolecular disulfide bridge, leaving no free sulfhydryl group to form dimers or oligomers.In leukocyte interferons (IFN-as), which contain four cysteine residues, there are two -S-S-bonds; between Cys-29 and Cys-138, and between Cys-1 and Cys-98 (7). Cys-141of IFN-p8 is required for biological activity (8). By analogy with the IFN-as, the Cys-141 of IFN-,B could be involved in an -S-S-bond with Cys-31, leavin...

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The purpose of this study was to determine whether human tumor cell lines derived from a single tumor exhibit heterogeneous responses to the anti-tumor effects of human recombinant tumor necrosis factor (h-r-TNF). Several cell lines with different metastatic propensities have been established in culture from a single surgical specimen of a human renal cell carcinoma following different selection procedures in nude mice. The cell lines exhibited significant differences in in vitro susceptibilities to cytotoxic effects of TNF. Kinetic analysis of the interaction of TNF with susceptible renal carcinoma cells demonstrated that a short 30 min interaction of TNF with the cells is sufficient to produce significant lysis 72 hr later. One cycle of exposure of sensitive cells to h-r-TNF did not produce cells resistant to it, nor did the degree of sensitivity to h-r-TNF vary after one passage of the cells in nude mice. Tumor cell resistance to the effects of TNF did develop spontaneously after prolonged cultivation in culture. We conclude that tumor cells derived from a single human renal cell carcinoma exhibit a heterogeneous response to the cytotoxic effects of h-r-TNF.

We investigated the binding of 125I-labeled beta interferon (IFN-beta Ser17), a nonglycosylated recombinant human fibroblast interferon in which cysteine at position 17 is replaced by serine by site-specific mutagenesis. An optimized chloramine T radiolabeling method produced a highly labeled, fully active 125I-IFN suitable for these studies. Unlike the case with the chloramine T method, incorporation of a single mole of Bolton-Hunter reagent into a mole of IFN-beta Ser17 led to nearly complete loss of biological activity. 125I-IFN-beta Ser17, prepared by the chloramine T method, bound specifically to human lymphoblastoid cells (Daudi) with a dissociation constant of 0.24 nM. The number of binding sites per cell was 4,000. In competition assays, unlabeled beta interferons (native, recombinant IFN-beta Cys17, and various preparations of IFN-beta Ser17) equally displaced labeled IFN-beta Ser17 on Daudi cells. Recombinant IFN-alpha-1 displaced 125I-IFN-beta binding to Daudi cells less efficiently than did unlabeled native or recombinant beta interferon. However, at the concentrations tested, native gamma interferon showed no competition with 125I-IFN. Our results indicate that IFN-beta Ser17 and native IFN-beta posses similar binding properties.