No abstract

Tumor necrosis factor (TNF) is cytotoxic for several transformed cell lines in vitro. In the presence of LiCI, the murine fibrosarcoma cell lines L929 and WEHI 164 done 13 became >10 times more sensitive to TNF-mediated cytotoxicity. The human tumor cell lines BT20 and HeLa D98/AH2 were also responsive to the cytotoxicity-enhancing effect of LiCI. Other monovalent or divalent cations did not affect TNF-mediated cytotoxicity. The potentiating effect of LiCI on TNF cytotoxicity was largely independent of transcription, and LiCl could be added to the cells as early as 2 hr before or as late as 4 hr after TNF without loss of effectiveness. The mechanism by which LiCI increases the cytotoxic response seems to differ from the sensitizing effect of actinomycin D or interferon y, since the latter treatments overcame TNF resistance of several cell lines, whereas LiCl did not. Evidence is presented that LiCl acts, either directly or indirectly, via the TNF-activated phospholipase A2 pathway. In nude mice, a combination ofTNF and LiCl led to hemorrhagic necrosis and growth inhibition of L929 tumors, whereas little effect was observed when TNF was administered alone. HeLa D98/AH2 tumors also were sensitive to the potentiating effect of LiCl in vivo. We conclude that LiCl enhances the effectiveness of TNF in vitro and in vivo, results that may have therapeutic implications.Tumor necrosis factor (TNF) is a cytokine that was originally identified in the sera of mice that had been primed with bacillus Calmette-Guerin and challenged with endotoxin. When injected into mice bearing methylcholanthreneinduced sarcomas, TNF causes hemorrhagic necrosis of the tumor (1). In vitro, TNF exerts cytostatic and cytotoxic activity against a wide range of human and murine tumor cell lines, although it has little or no antiproliferative activity on nontransformed cell lines (2, 3). However, not all tumor cells are sensitive to TNF-mediated cytotoxicity. The molecular mechanism for this difference in response still remains largely unknown. Recently, a serine-type protease was shown to be involved in TNF-mediated cytotoxicity (4). Furthermore, there is ample evidence for an activation of a phospholipase A2 (PLA2) activity (5, 6). In addition to its cytotoxic effect on transformed cells, TNF mediates a variety of other biological activities on various cell types, both in vivo and in vitro (7).In our efforts to understand the mechanism of action of TNF on malignant cells, we also evaluated a possible involvement of phospholipase C activity. In these experiments, we tested whether LiCl, which is known to inhibit inositol-1-phosphatase (8), would interfere with specific tumor cell killing by TNF. Surprisingly, we found instead that LiCl potentiated TNF-mediated cytotoxicity in vitro almost to a similar extent as has been shown before for actinomycin D (ActD) and cycloheximide (2) and, more physiologically, for interferon (IFN) (3, 9). Moreover, this LiCl-specific TNF potentiation can be extended to the in vivo antitumor action of TNF. Elsewh...

L929, a murine fibrosarcoma cell line highly sensitive to the anti-proliferative and cytotoxic action of tumour necrosis factor (TNF), was used as a target cell in our studies. We [Suffys et al. (1987) Biochem. Biophys. Res. Commun. 149, 735 -7431, as well as others, have previously provided evidence that a phospholipase (PL), most probably a PL-A,-type enzyme, is likely to be involved in TNF-mediated cell killing. We now further document this conclusion and provide suggestive evidence that the enzyme activity specifically involved in TNF cytotoxicity differs from activities associated with the eventual cell death process itself or with non-toxic serum treatment. We also show that the 5,8,11,14-icosatetraenoic acid (arachidonic acid, A4Ach) released by PL, and possibly metabolized, is unlikely to be a key mediator of the TNF-mediated cytotoxicity. These conclusions are based on the following experimental findings.1. TNF treatment of cells, prelabelled for 24 h with [jH]A4Ach or [14C]A3Ach (A3Ach = 5,8,11-icosatrienoic acid) resulted in an early, time-dependent and concentration-dependent release of radioactivity in the supernatant preceding actual cell death. The extent of this response was moderate, albeit reproducible and significant. Analysis of the total lipid fraction from cells plus supernatant revealed that only release of arachidonic acid from phospholipids, but not its metabolization was induced by TNF. However, the release of less unsaturated fatty acids, such as linoleic acid (Lin) or palmitic acid (Pam), was not affected during the first hours after TNF addition.2. An L929 subclone, selected for resistance to TNF toxicity, was found to be defective in TNF-induced A4Ach liberation.3. Interleukin-1 (IL1) was not cytotoxic for L929 and did not induce release of A4Ach. 4. Release of A4Ach was not restricted to TNF; the addition of serum to the cells also induced release of fatty acids into the medium. In this case, however, there was no specificity, as all fatty acids tested, including Lin and Pam, were released.5. Inhibition of PL-A, activity by appropriate drugs markedly diminished TNF-induced A4Ach release and resulted also in a strong decrease in TNF-induced cytotoxicity.6. Other drugs, including serine protease inhibitors, which strongly inhibit TNF-induced cytotoxicity, also decreased the TNF-induced A,Ach release, whereas LiCl potentiated both TNF-mediated effects.7. Protection of cells against TNF toxicity by means of various inhibitors was not counteracted by addition of exogenous fatty acids, including A,Ach.8. We could not detect an increase in the amount of free inositol or any inositol phosphate after TNF treatment of cells, prelabelled with my~- [~H]inositol. This indicates that the phosphatidylinositol-specific PL-C is not involved in the TNF-induced fatty acid release. 9. Neither were we able to observe a decrease in fatty acid incorporation into phospholipids during TNF treatment. This excludes a decrease in acyltransferase activity as the reason for increased levels of free fatty acid...

We investigated the effect of various protease inhibitors on the anti-proliferative and cytotoxic action of tumour necrosis factor (TNF) on mouse L929 fibrosarcoma cells.1. The following serine-type protease inhibitors led to inhibition of TNF action: phenylmethylsulfonyl fluoride, N"-p-tosyl-L-lysine chloromethane, Nu-p-tosyl-L-phenylalanylchloromethane, Na-p-tosyl-L-arginine methyl ester, L-leucine methyl ester, DL-phenylalanine methyl ester, N-acetyl-DL-phenylalanine-8-naphthyl ester, p-nitrophenyl p'-guanidino-benzoate and antipain. We could not detect an effect of inhibitors specific for thiol protease on TNF.2. Inhibition of TNF-mediated cytotoxicity was evident in both the presence and absence of actinomycin D or cycloheximide.

Tumor necrosis factor (TNF) and interleukin 1 (IL-l) are both cytokines of macrophage origin with similar activity on several cell types. We investigated whether TNF can, analogously to IL-l, stimulate phospholipase activity of chondrocytes. Addition of each of these cytokines to cells, isolated from the xiphistemum of adult rats, resulted in a time-and dose-dependent increase in phospholipase activity in both secreted and membrane-associated form. Moreover, TNF and IL-1 both induce a transformation of chondrocyte morphology. In conclusion, TNF stimulates chondrocyte phospholipase activity and extends the long list of actions shared by IL-l and TNF in a diversity of cellular systems.Tumor necrosis factor; Interleukin 1; Phospholipase; (Rat chondrocyte)