Biomedical subjects

B Vanhaesebroeck

Publications and source records attributed to B Vanhaesebroeck.

46 records · Page 3Linked to original sources

PubMed1991

Expression of the tumor necrosis factor gene in tumor cells correlates with reduced tumorigenicity and reduced invasiveness in vivo.

We investigated whether a constitutive production of low amounts of tumor necrosis factor (TNF) by neoplastic cells affects their in vivo tumorigenicity. TNF-resistant derivatives were isolated from the TNF-sensitive murine fibrosarcoma cell lines L929s and WEHI164cl13s, L929r1-type TNF-resistant subclones were found to constitutively produce TNF in vitro, in contrast to non-TNF-producing but TNF-resistant L929r2 and WEHI164cl13r2 cell clones. The TNF-sensitive parental cell lines as well as the L929r2 and WEHI164cl13r2 cell lines similarly induced fast-growing tumors upon s.c. inoculation into nude mice (Swiss-nu/nu). In contrast, the TNF-producing L929r1-type cells showed reduced tumorigenicity and in vivo growth rate, which both inversely correlated with the level of in vitro-produced TNF. Tumor take incidence but not the in vivo growth rate of L929r1-type cells was greatly facilitated by prior whole body gamma-irradiation (350 rads) of the recipient, implying the involvement of host mechanisms at least in the lower take incidence of L929r1 tumors. These host mechanisms, possibly activated by tumor-produced TNF, acted only locally, inasmuch as the growth of an inoculum of L929s cells was not influenced either by a simultaneous distant inoculum of L929r1 cells or by established, distant L929r1 tumors. Efforts to eliminate these host mechanisms by prior local UV irradiation of the skin were unsuccessful. All L929 cell types were found to be similarly susceptible to killing by host cytotoxic effector cells (macrophages and natural and lymphokine-activated killer cells). Histological investigation did not reveal clear differences in tumor-associated inflammatory cells but revealed that tumors induced by L929r1-type cells, in contrast to L929s and L929r2 tumors, did not show invasiveness in host tissues. Moreover, L929r1 tumors were frequently encapsulated, which was never observed for tumors induced by L929s and L929r2 cells. Taken together, our results suggest that tumor-derived TNF locally activates host antitumor activities. Possible effector mechanisms are discussed.

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

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

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↗

PubMed1990

Modulation of cellular susceptibility to the cytotoxic/cytostatic action of tumor necrosis factor by adenovirus E1 gene expression is cell type-dependent.

Primary baby rat kidney cells, primary human embryonic retinoblast cells, established NIH3T3 and established normal rat kidney (NRK) cells, expressing E1A and/or E1B gene regions of adenovirus 5 (Ad5) or Ad12, were investigated for susceptibility to the cytotoxic/cytostatic action of Tumor Necrosis Factor (TNF). In the primary cells and in the NRK cells, there was no correlation between TNF sensitivity and E1 gene expression; neither did sensitivity to TNF correlate with the oncogenicity of the Ad serotype. In contrast, the expression of Ad E1 gene regions in NIH3T3 cells was found to enhance TNF sensitivity of this cell line. Differences in E1A expression levels between cell types cannot explain this discrepancy regarding modulation of TNF sensitivity by E1A.

Adenovirus Early Proteins↗

PubMed1989

An ongoing in vivo immune response affects the abundancy and differentiation of lymphokine-activated killer cell precursors, but does not influence their broad spectrum target reactivity.

Using a model of local lymph node (LN) immunization, we investigated the effect of in vivo Ir on the generation of lymphokine-activated killer (LAK) cells or their precursors. Ag used for immunization were SRBC, horse RBC, OVA, keyhole limpet hemocyanin, or CFA. Ag-draining LN, in the acute phase of the Ir, did not contain detectable LAK effector activity, nor an enhanced NK activity. After culture for 3 to 5 days in the absence of exogenously added IL-2, immunized LN cells developed a spontaneous LAK-like cytotoxicity. This activity represented a substantial fraction of the IL-2-generated LAK cytolysis and was mediated by a Thy-1+ cell population phenotypically indistinct from IL-2-induced LAK. Inclusion (on day 0 of culture) of antibodies to IL-2, IL-2R, IL-4, IL-6, IFN-gamma, or TNF suggests a marginal involvement of IL-2 and IL-4 in the generation of this response. LAK, induced in vitro by exogenously added IL-2, developed earlier in LN cells immunized with particle Ag (SRBC, horse RBC, and CFA), but not with protein Ag (OVA and keyhole limpet hemocyanin). This effect was not mediated by endogenous IL-4. During further culture time in the presence of a saturating IL-2 concentration, similar levels of LAK activity were generated in naive and immunized LN cells. This agrees with the similar or slightly higher LAK precursor frequencies in immunized versus naive LN as assessed by limiting dilution experiments. Considering the 2.7-fold to 18-fold increase in cell content of the immunized LN, due to a recruitment and expansion of Ag-reactive B and T lymphocytes, a de novo generation of LAK precursors at the site of the Ir, and resulting from the Ir, must be assumed. In conclusion, our results suggest an interrelation between immune reactivity and LAK responses.

Animals↗

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

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

Structure-function relationship of tumour necrosis factor and its mechanism of action.

We have cloned the cDNAs of both human and mouse TNF and expressed them to high efficiency in Escherichia coli. Many transformed cell lines are sensitive to the cytotoxic action of TNF, especially in the presence of gamma-interferon, whereas normal cells either are unaffected or respond mitogenically. A number of human-mouse chimeric TNF genes have been constructed and expressed. All show biological activity but none of the chimeric proteins is neutralized by monoclonal antibodies to TNF. TNF has potent antitumour activity in nude mice carrying human xenografts or in mice bearing syngeneic tumours. In some systems direct effects can be demonstrated (in combination with species-specific gamma-interferon) but in others TNF acts indirectly. Combination of TNF with cytostatic drugs can also be effective in curing in vivo. The major limitation of the use of TNF is its toxicity. On many cell types TNF has an action similar to interleukin 1 (IL-1). At least some of the secondary, intracellular events may be identical for the two effectors. A possible mechanism of action of TNF is the release and metabolism of polyunsaturated fatty acids, which would explain the synthesis of prostaglandins and leukotrienes by many cell types after TNF treatment. The activation of the phospholipase can be blocked by corticoids. Some protease inhibitors protect cells from TNF-induced cytotoxicity but the target of these inhibitors has not been identified. Several genes are switched on by TNF (and by IL-1), including the gene for the 26 kDa protein recently identified as B cell stimulation factor 2. Events preceding death in rats include hypothermia, hypotension, acidosis and hypoglycaemia. All these effects can be largely eliminated by indomethacin pretreatment, with a resulting improvement in survival. As indomethacin does not inhibit the cytotoxic action of TNF on malignant cells it may form the basis for improved treatment protocols.

Animals↗

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↗