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R Beyaert

Publications and source records attributed to R Beyaert.

At least 73 records · Page 4Linked to original sources
PubMed1993

Effect of bcl-2 proto-oncogene expression on cellular sensitivity to tumor necrosis factor-mediated cytotoxicity.

Introduction and expression of the proto-oncogene bcl-2 (B-cell lymphoma/leukemia 2) has been shown to extend the survival of certain hematopoietic cell lines after growth factor deprivation, by blocking apoptosis or programmed cell death. We investigated the effect of bcl-2 expression on cellular sensitivity to lysis by tumor necrosis factor (TNF), a cytokine capable of inducing apoptosis in several tumor cell lines. Introduction of the human bcl-2 gene in the highly TNF-sensitive L929 mouse fibrosarcoma cell line did not result in altered TNF sensitivity. Likewise, NIH3T3 and REF cells, which are resistant to TNF cytotoxicity but become TNF sensitive upon cotreatment with actinomycin D or upon expression of the adenovirus E1A gene, did not show altered TNF sensitivity upon bcl-2 transfection. Despite constitutive expression of the endogenous bcl-2 gene, human MCF7 breast carcinoma cells, as well as HL60 promyelocytic leukemia and U937 histiocytic lymphoma cell lines were found to be TNF sensitive. bcl-2-overexpressing derivatives of these cell lines did not acquire reduced TNF sensitivity and still exhibited the characteristic pattern of internucleosomal DNA fragmentation of TNF-induced apoptosis. Moreover, bcl-2 expression in the interleukin 3 (IL-3)-dependent myeloid cell line 32D protected these cells from apoptosis resulting from growth factor deprivation, but not from apoptosis induced by TNF. These data clearly establish the absence of a correlation between bcl-2 gene expression and cellular sensitivity to TNF-induced cell lysis. These findings are discussed in the context of the hypothesis of different pathways for induction of apoptosis, only some of which are affected by bcl-2 expression.

3T3 Cells↗
PubMed1992

Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation.

Structural mitochondrial damage accompanies the cytotoxic effects of several drugs including tumor necrosis factor (TNF). Using various inhibitors of mitochondrial electron transport we have investigated the mechanism of TNF-mediated cytotoxicity in L929 and WEHI 164 clone 13 mouse fibrosarcoma cells. Inhibitors with different sites of action modulated TNF cytotoxicity, however, with contrasting effects on final cell viability. Inhibition of mitochondrial electron transport at complex III (cytochrome c reductase) by antimycin A resulted in a marked potentiation of TNF-mediated injury. In contrast, when the electron flow to ubiquinone was blocked, either at complex I (NADH-ubiquinone oxidoreductase) with amytal or at complex II (succinate-ubiquinone reductase) with thenoyltrifluoroacetone, cells were markedly protected against TNF cytotoxicity. Neither uncouplers nor inhibitors of oxidative phosphorylation nor complex IV (cytochrome c oxidase) inhibitors significantly interfered with TNF-mediated effects, ruling out the involvement of energy-coupled phenomena. In addition, the toxic effects of TNF were counteracted by the addition of antioxidants and iron chelators. Furthermore, we analyzed the direct effect of TNF on mitochondrial morphology and functions. Treatment of L929 cells with TNF led to an early degeneration of the mitochondrial ultrastructure without any pronounced damage of other cellular organelles. Analysis of the mitochondrial electron flow revealed that TNF treatment led to a rapid inhibition of the mitochondria to oxidize succinate and NADH-linked substrates. The inhibition of electron transport was dose-dependent and became readily detectable 60 min after the start of TNF treatment, thus preceding the onset of cell death by at least 3-6 h. In contrast, only minor effects were observed on complex IV activity. The different effects observed with the mitochondrial respiratory chain inhibitors provide suggestive evidence that mitochondrial production of oxygen radicals mainly generated at the ubisemiquinone site is a causal mechanism of TNF cytotoxicity. This conclusion is further supported by the protective effect of antioxidants as well as the selective pattern of damage of mitochondrial chain components and characteristic alterations of the mitochondrial ultrastructure.

Amobarbital↗
PubMed1992

Synergistic induction of interleukin-6 by tumor necrosis factor and lithium chloride in mice: possible role in the triggering and exacerbation of psoriasis by lithium treatment.

One of the side effects of treatment of manic depressive disease with lithium salts is the triggering or aggravation of psoriasis. In a murine model, subcutaneous (s.c.) injection of a combination of tumor necrosis factor (TNF) and lithium chloride (LiCl) induces a psoriasiform inflammatory reaction. Recent studies suggest that interleukin (IL)-6 and its inducer TNF may play an important role in the pathophysiology of psoriasis. To understand the mechanism involved in the exacerbation of psoriasis by lithium salts, the IL-1, IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels in murine skin injected with TNF in combination with LiCl were studied. IL-6 levels in skin extracts of mice treated s.c. with a combination of TNF and LiCl were considerably increased as compared to the levels found in skin extracts from mice treated with TNF or LiCl alone. In contrast, in the same skin extracts IL-1 levels were not changed and GM-CSF was even not detectable. Although less pronounced, increased IL-6 levels could also be found in the sera of mice treated s.c. with TNF and LiCl. Injection with IL-1, interferon-gamma, lipopolysaccharide, or phorbol 12-myristate 13-acetate also induced IL-6 in murine skin. However, these IL-6 levels were not enhanced by co-treatment with LiCl. Likewise, on inflammatory reaction could be seen in mice treated with these agents. These results suggest a role for endogenous TNF and IL-6 in the triggering or aggravation of psoriasis in lithium-treated patients.

Animals↗
PubMed1991

Two discrete types of tumor necrosis factor-resistant cells derived from the same cell line.

From the murine fibrosarcoma cell line L929s, which is sensitive to tumor necrosis factor (TNF)-mediated cell lysis, two discrete types of TNF-resistant variants were derived by TNF selection. Cells of the first type (named L929r1) were not sensitized to TNF cytotoxicity by cotreatment with either inhibitors of protein or RNA synthesis, or gamma-interferon, despite the presence of a functional gamma-interferon response. L929r1 constitutively produced TNF in the supernatant and expressed membrane-bound TNF, which was not bound to the TNF receptor. In fact, TNF receptors could not be demonstrated on L929r1 cells, not even after low pH treatment and/or incubation with antiserum to TNF. L929r1 exhibited a stable TNF-resistant phenotype in the absence of further TNF selection. No evidence could be obtained that TNF acted as an autocrine growth factor for these cells. L929r2, the second type of TNF-resistant L929 cells, became sensitive to TNF lysis in the presence of RNA or protein synthesis inhibitors, or in the presence of gamma-interferon. TNF induced the secretion of interleukin 6 in these cells, additionally showing that functional TNF signaling in these cells indeed takes place, but does not lead to cell lysis under normal conditions. L929r2 did not produce TNF, also not upon stimulation with exogenous TNF. The number and binding affinity of TNF receptors were not consistently different between L929s and L929r2 cells. In the absence of further TNF selection, L929r2 gradually reverted to TNF sensitivity. This sensitivity was not reversible to TNF resistance by the gene-regulatory agents 5-azacytidine or sodium butyrate. Treatment with these agents also did not affect the TNF sensitivity of L929s cells nor the TNF resistance of L929r1 and L929r2 cells. In summary, our results suggest the existence among cells of the same cell line of discrete mechanisms for acquisition of resistance to TNF-mediated cell lysis.

Animals↗
PubMed1991

Tumour-necrosis-factor-mediated cytotoxicity is correlated with phospholipase-A2 activity, but not with arachidonic acid release per se.

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, 735-743], as well as others, have previously provided evidence that a phospholipase (PL), most probably a PL-A2-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, delta 4 Ach) 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 [3H] delta 4Ach or [14C] delta 3Ach (delta 3Ach identical to 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 delta 4Ach libration. 3. Interleukin-1 (IL1) was not cytotoxic for L929 and did not induce release of delta 4Ach. 4. Release of delta 4Ach 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-A2 activity by appropriate drugs markedly diminished TNF-induced delta 4Ach 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 delta 4Ach 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 delta 4Ach.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Lithium chloride potentiates tumor necrosis factor-induced and interleukin 1-induced cytokine and cytokine receptor expression.

The antimalignant cell activity of tumor necrosis factor (TNF) in many cell types can be enhanced by lithium chloride (LiCl). This study shows the in vitro effect of LiCl on the TNF-induced or interleukin 1 (IL-1)-induced expression of IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, IL-2, and the IL-2 receptor-alpha (IL-2R alpha). The levels of IL-6 and GM-CSF in the medium of TNF-treated L929 fibrosarcoma cells were increased by cotreatment with LiCl. In contrast, enhancement of IL-6 production by dibutyryl cyclic AMP or cycloheximide was not affected by LiCl. The production of IL-6 and GM-CSF was not correlated with sensitivity to TNF-mediated cell killing. IL-1 by itself had no measurable effects on L929 cells. However, LiCl potentiated the IL-1-induced synthesis of IL-6, GM-CSF, IL-3, and IL-2 in PC60 murine T-cell hybridoma cells. TNF alone induced only GM-CSF production in these cells, but in the presence of LiCl, increased amounts of GM-CSF as well as small amounts of IL-2 and IL-6 could be detected. It is also shown that in these PC60 cells the expression of the IL-2R alpha was induced by TNF + LiCl treatment but not by TNF alone. IL-2R alpha expression was likewise considerably enhanced by IL-1 + LiCl treatment, as compared with treatment with IL-1 alone. The effects of LiCl on the TNF-induced and the IL-1-induced gene expression seem to be independent of the protein kinase A and C pathways. These results show that LiCl can modulate both TNF-mediated cytotoxicity and TNF-induced and IL-1-induced cytokine expression, suggesting that Li+ acts early in the TNF-signaling pathway, but at a step shared with the IL-1-signaling pathway.

Animals↗
PubMed1991

Induction of inflammatory cell infiltration and necrosis in normal mouse skin by the combined treatment of tumor necrosis factor and lithium chloride.

Previously we reported that lithium chloride (LiCl) potentiates tumor necrosis factor (TNF)-mediated cytotoxicity in vitro and in vivo. Here, using a murine normal skin model, it is shown that a subcutaneous injection of TNF plus LiCl induces acute dermal and subcutaneous inflammation and necrosis. Histology showed a marked initial dermal and subcutaneous neutrophil infiltrate by approximately 2 hours, followed by a predominantly mononuclear infiltrate by 24 hours, which remained present for several days. Tumor necrosis factor or LiCl alone induced negligible inflammation, disappearing after 6 hours; furthermore there was never necrosis or ulceration of the overlying skin in case of single-agent application. In vitro studies showed that the combination of TNF and LiCl, but not either agent alone, was directly cytotoxic to fibroblastic cells of murine skin. No inflammatory infiltration was visible in tumors treated intratumorally or perilesionally with TNF plus LiCl, although the latter treatment resulted in a perilesional leukocyte infiltration. Furthermore the combination of TNF and LiCl had no effect on macrophage cytotoxicity to L929 tumors.

Animals↗
PubMed1990

Hormone sensitivity in vitro and in vivo of v-ras-transfected MCF-7 cell derivatives.

Human mammary carcinoma cell lines (MCF-7) were analysed for their hormone sensitivity before and after transfection with a v-Ha-ras oncogene or with a neomycin-resistance gene followed by selection in vitro or in vivo. Our aim was to test how the expression of the ras oncogene would influence the estradiol sensitivity of MCF-7 cells. In culture, MCF-7 cells expressing the viral p21 oncogene product, as compared to parental MCF-7 cells and their control derivatives, showed lower levels of a 67-kDa estrogen receptor. Progesterone receptors, however, remained sensitive to up-regulation by estrogens. The oncogene-expressing cells were less sensitive than all controls to stimulation of proliferation by 10(-8)M estradiol or to inhibition of proliferation by 2-CH3-4-OH tamoxifen, and this was not dependent upon the type of culture medium used. After s.c. or i.p. injection into female athymic nude mice, ovariectomized or left intact, the growth of MCF-7 cells expressing the ras oncogene product and of all control cells was sensitive to stimulation by estrogen supplementation. Conversely, cell lines derived from tumors generated with long latency in untreated athymic nude mice by v-ras-expressing MCF-7 cells showed efficient formation of quickly growing tumors in the absence of estrogen supplementation. No differences were observed in invasion and metastasis of the different MCF-7 cell types injected into athymic nude mice that were supplemented with estrogens or not.

Animals↗
PubMed1990

Inhibition by glucocorticoids of tumor necrosis factor-mediated cytotoxicity. Evidence against lipocortin involvement.

The role of the phospholipase inhibitor proteins, lipocortin-I and -II, in tumor necrosis factor (TNF)-mediated cytotoxicity against L929 fibrosarcoma cells was investigated. We previously reported that TNF-mediated cytotoxicity was inhibited by dexamethasone (DEX), suggesting an involvement of lipocortins. Now we show that, despite inhibition by DEX of TNF-induced arachidonic acid release, DEX has no effect on the synthesis of these lipocortins. Moreover, TNF itself has no effect on the synthesis and phosphorylation of lipocortin-I and -II. Also there was no difference in expression levels of lipocortin-I and -II between TNF-sensitive and -resistant cells. These data strongly suggest that the protective effect of DEX and other glucocorticoids is not mediated by lipocortins.

Annexins↗
PubMed1990

Cytotoxic activity of tumor necrosis factor is inhibited by amiloride derivatives without involvement of the Na+/H+ antiporter.

Cytotoxicity of tumor necrosis factor (TNF) on L929s cells was efficiently blocked by several amiloride analogs but not by amiloride itself. This protection did not require RNA or protein synthesis. Na+/H+ antiporter-negative L-M(TK-) cells (LAP) could be killed by TNF, showing that the Na+/H+ exchanger is not required for TNF-cytotoxicity. Similar protection against TNF-mediated cell lysis by amiloride derivatives was found for LAP and L929s cells, excluding a blockade of the Na+/H+ antiporter as the cause of the protection against TNF by these agents.

Amiloride↗
PubMed1989

Lithium chloride potentiates tumor necrosis factor-mediated cytotoxicity in vitro and in vivo.

Tumor necrosis factor (TNF) is cytotoxic for several transformed cell lines in vitro. In the presence of LiCl, the murine fibrosarcoma cell lines L929 and WEHI 164 clone 13 became greater than 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 LiCl. Other monovalent or divalent cations did not affect TNF-mediated cytotoxicity. The potentiating effect of LiCl 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 LiCl increases the cytotoxic response seems to differ from the sensitizing effect of actinomycin D or interferon gamma, 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 of TNF 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.

Animals↗
PubMed1988

Involvement of a serine protease in tumour-necrosis-factor-mediated cytotoxicity.

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 alpha-p-tosyl-L-lysine chloromethane, N alpha-p-tosyl-L-phenylalanyl chloromethane, N alpha-p-tosyl-L-arginine methyl ester, L-leucine methyl ester, DL-phenylalanine methyl ester, N-acetyl-DL-phenylalanine-beta-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. 3. TNF itself was not found to be a protease, as it had no proteolytic activity in a sensitive colorimetric assay. [1,3-3H]Diisopropyl fluorophosphate, an effective irreversible inhibitor of serine proteases, did not bind to TNF. Pretreatment of TNF with N alpha-p-tosyl-L-lysine chloromethane did not influence its biological activity. 4. The addition of protease inhibitor to the cells at various times after TNF administration led to a gradual loss of protection, suggesting that the protease acts at a rather late stage. 5. Protease inhibitors did not influence TNF binding, internalization or metabolization. 6. No increase in supernatant protease activity or in cell-associated protease activity could be detected after treatment of L929 cells with TNF. Our results document the involvement of protease activity, acting quite late during the cytolytic and growth inhibiting processes induced by TNF.

Animals↗
PubMed1988

TNF: its potential as an antitumour agent.

Tumour Necrosis Factor (TNF) was discovered on the basis of its capability to induce necrosis of certain tumours in vivo. A brief overview is given of the pleiotropic effects of TNF on a variety of cells, either transformed cells or normal, diploid cells. Many transformed cells are killed by TNF, especially in the presence of interferon-gamma or inhibitors of transcription or translation. Various activities of TNF on normal cells have been studied, especially those on the endothelial system; these effects may be relevant to an understanding of its toxicity. TNF presumably acts by activation of phospholipase-A2. A number of genes are induced by TNF and, for example, many cells produce interleukin-6. The latter acts on B-cells, on T-cells, on bone marrow cells and, last but not least, on hepatocytes, which results in the synthesis of acute phase proteins. Although the toxicity of TNF, especially in the presence of interferon, limits its wide applicability, it can nevertheless lead to complete tumour curing in experimental animals. Reduction of its toxicity, e.g. by indomethacin treatment, opens new possibilities for TNF as an antitumour drug, alone or in combination with interferon.

Animals↗
PubMed1987

Reduced tumour necrosis factor-induced cytotoxicity by inhibitors of the arachidonic acid metabolism.

The mechanism of tumour necrosis factor-mediated cytotoxicity was investigated by using various inhibitors of arachidonic acid metabolism. Phospholipase A2 inhibitors with different modes of action interfered with the cytotoxic action of TNF, whereas phospholipase C inhibitors did not. Neither cyclooxygenase nor lipoxygenase-blockers had a significant effect on TNF action. Experiments with scavengers of toxic oxygen radicals gave ambiguous results. The data obtained suggest the involvement of phospholipase A2 and arachidonic acid in the cytotoxic mechanism of TNF, but the exact role of these molecules is, however, still to be determined.

Arachidonic Acid↗
PubMed1987

Gene cloning and structure--function relationship of cytokines such as TNF and interleukins.

The genes for a number of proteins, potentially useful in cancer therapy and collectively called "biological response modifiers", have been cloned and expressed in micro-organisms in recent years. These recombinant proteins, which are now available in pure form in nearly unlimited quantities, include interferons, interleukins and cytotoxins such as Tumor Necrosis Factor (TNF) and lymphotoxin. Most often the human gene has been cloned and expressed, with view to possible applications in medicine, but usually the mouse equivalent gene was also characterized in order to carry out syngeneic animal model experiments. TNF is selectively toxic for many transformed cell lines, either alone or in combination with interferon or inhibitors of RNA or protein synthesis. Cells sensitive to the cytotoxic action of TNF and cells unaffected by it nonetheless usually carry about an equal number of TNF receptors; hence it is the secondary, intracellular signal which makes the difference between a transformed cell and a normal, diploid cell. TNF can induce a number of different genes in a variety of cells; for example, endothelial cells express a surface antigen responsible for adherence of leucocytes. Another gene which is induced by TNF is interleukin 6 (also called 26 kDa protein or BSF-2). This interleukin, IL-6, is a growth and differentiation factor for B cells as well as for T cells; it is responsible for functions previously ascribed to hepatocyte-stimulating factor, but has no interferon activity. The toxic action of TNF on tumor cells must involve the release of arachidonic acid as phospholipase inhibitors block the TNF-induced effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Inhibition of tumor necrosis factor-induced necrotic cell death by the zinc finger protein A20.

Tumor Necrosis Factor (TNF) is a cytokine that induces necrotic and apoptotic forms of cell death. The TNF-induced signalling mechanisms leading to necrosis or apoptosis are partially distinct, and are therefore likely to be regulated in a different way. The zinc finger protein A20 is a TNF-induced primary response gene that has been shown to inhibit TNF-induced apoptosis. However, its ability to inhibit the necrotic route of cell death as well as the underlying mechanism remains unknown. Here we show that stable expression of A20 or a fusion protein consisting of Green Fluorescent Protein (GFP) and A20 protects the TNF-sensitive fibroblast cell line L929 partially from TNF-induced necrotic cell death. TNF-induced necrosis has been shown to involve the activation of several phospholipases, as well as an increased production of reactive oxygen radicals. The reduced TNF-sensitivity of A20-expressing L929 cells was correlated with a decrease of TNF-induced phospholipase A2 (PLA2), phospholipase C (PLC) and phospholipase D (PLD) activation. Furthermore, production of mitochondrial reactive oxygen intermediates was retarded by overexpression of A20. These results demonstrate that A20 not only inhibits TNF-induced apoptosis but also TNF-induced necrosis, suggesting that it interferes with an early step in TNF signalling which is required for both types of cell death.

Animals↗