Purification and characterization of a human tumor necrosis factor from the LuKII cell line.

Abstract

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. Purified human TNF(LuKII) (i) produces hemorrhagic necrosis of Meth A mouse sarcoma in the standard in vivo mouse TNF assay; (ii) has the same pattern of reactivity as mouse TNF (cytotoxic/cytostatic/no effect) on a panel of human cancer cell lines; and (iii) has its anticellular effect potentiated by interferon, also a feature of mouse TNF.

Sendai virus induces high levels of tumor necrosis factor mRNA in human peripheral blood leukocytes

“…As the cytotoxin TNF was originally named for a protein produced in erndotoxin-treated mice (1), most studies of TNF have focused on induction by endotoxin, although certain mitogens and phorbol myristate acetate have also been shown to induce TNF (34). Even though Rmultiple closely-related TNFs may exist (4,17), the deduced amino acid sequences for mature TNF cloned from the tumor-derived HL-60 (3, 4) and U-937 (6) cell lines and lung macrophages (7) A recent study (36) has also shown that Sendai virus induces peripheral blood mononuclear cells to express a cytotoxin, which is believed to be TNF on the basis of neutralization of cytotoxic activity with a recombinant TNF n,onoclonal antibody. That report, however, may offer evidence for the existence of a cytotoxin closely-related to the cloned TNF, since the majority of the cytotoxic activity in that study eluted on a gel filtration column with a relative molecular mass of approximately 14,000 daltons.…”

“…Cytotoxins produced by the immurne system are a heterogeneous group of proteins, the nature of which depends upon the variety of inducing agent and target cell involved (16)(17)(18)33). As the cytotoxin TNF was originally named for a protein produced in erndotoxin-treated mice (1), most studies of TNF have focused on induction by endotoxin, although certain mitogens and phorbol myristate acetate have also been shown to induce TNF (34).…”

“…Despite this, cytotoxic activity in the culture medium was at a low level. Since the previously cloned TNF was generated from endotoxin and phorbol myristate acetate induced tumor-derived cell lines, and since multiple cytotoxic TNF-like proteins may exist (4, [16][17][18], it was necessary to determine whether Sendai virus, a previously unknown inducer of TNF, was directing normal human cells to express TNF and whether the amino acid sequence of the expressed protein was identical to that of the previously cloned TNF. We compare the temporal expression of TNF mRNA and secreted cytotoxic activity to interferon-a (IFN-a) mRNA Oligonucleotides were synthesized using phosphoramidite chemistry with a Systec Microsyn 1450 synthesizer.…”

Natural human tumor necrosis factor beta (lymphotoxin) Variable O‐glycosylation at Thr7, proteolytic processing, and allelic variation

“…1, lanes 3,4,6,7). TNF-/3 components with high molecular weight observed by others working with iodilmted protein [12] thus most likely also represent artefacts of the preparation.…”

“…)tt results, with molecular masses varying between 20 and 70 kDa [2,10,11]. In several studies, heterogeneous preparations were obtained, with up to seven bands with apparent masses between 19 and 80 kDa [12]. Various groups, using radiolabelled preparations, provided evidence that TNF-fl is N-glycosylated and possibly also O-glycosylated [13-i5].…”