Biomedical subjects

B Vanhaesebroeck

Publications and source records attributed to B Vanhaesebroeck.

At least 37 records · Page 2Linked to original sources

PubMed1996

Structural and functional diversity of phosphoinositide 3-kinases.

Phosphoinositide 3-kinases (PI3-kinases) have been shown to be recruited to cell surface receptor signal complexes whose formation is triggered by growth factors, cytokines and other ligands. PI3-kinases are also involved in protein sorting phenomena. A number of PI3-kinase isotypes have been characterised in several laboratories. Here the relations between the PI3-kinases, PI4-kinases and PI5-kinases and other potential phosphoinositide kinases are analysed. A study of the relation of structure to function for sequence motifs defined through the use of homology searches and protein modelling techniques is described and used to assign the family of phosphoinositide kinases to subgroups.

Amino Acid Sequence↗

PubMed1996

Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction.

Wortmannin at nanomolar concentrations is a potent and specific inhibitor of phosphoinositide (PI) 3-kinase and has been used extensively to demonstrate the role of this enzyme in diverse signal transduction processes. At higher concentrations, wortmannin inhibits the ataxia telangiectasia gene (ATM)-related DNA-dependent protein kinase (DNA-PKcs). We report here the identification of the site of interaction of wortmannin on the catalytic subunit of PI 3-kinase, p110alpha. At physiological pH (6.5 to 8) wortmannin reacted specifically with p110alpha. Phosphatidylinositol-4,5-diphosphate, ATP, and ATP analogs [adenine and 5'-(4-fluorosulfonylbenzoyl)adenine] competed effectively with wortmannin, while substances containing nucleophilic amino acid side chain functions had no effect at the same concentrations. This suggests that the wortmannin target site is localized in proximity to the substrate-binding site and that residues involved in wortmannin binding have an increased nucleophilicity because of their protein environment. Proteolytic fragments of wortmannin-treated, recombinant p110alpha were mapped with anti-wortmannin and anti-p110alpha peptide antibodies, thus limiting the target site within a 10-kDa fragment, colocalizing with the ATP-binding site. Site-directed mutagenesis of all candidate residues within this region showed that only the conservative Lys-802-to-Arg mutation abolished wortmannin binding. Inhibition of PI 3-kinase occurs, therefore, by the formation of an enamine following the attack of Lys-802 on the furan ring (at C-20) of wortmannin. The Lys-802-to-Arg mutant was also unable to bind FSBA and was catalytically inactive in lipid and protein kinase assays, indicating a crucial role for Lys-802 in the phosphotransfer reaction. In contrast, an Arg-916-to-Pro mutation abolished the catalytic activity whereas covalent wortmannin binding remained intact. Our results provide the basis for the design of novel and specific inhibitors of an enzyme family, including PI kinases and ATM-related genes, that play a central role in many physiological processes.

Adenosine Triphosphate↗

PubMed1996

The study of phosphoinositide 3-kinase function.

Phosphoinositide 3-kinases catalyze the addition of phosphate at the 3-OH position of inositol lipids in cellular membranes. Initially identified as an enzymatic activity associated with transforming oncogenic proteins, PI3-kinases are now known to be involved in signal transduction through receptors with intrinsic or associated tyrosine kinase activity (such as sre type tyrosine kinases) and receptors linked to heterotrimeric G proteins. PI3-kinases also have a role in vesicular trafficking events. PI3-kinases form a large family of related proteins, conserved in organisms as distant as yeast and man. The exact role of their lipid products remains to be established, but the multiplicity of cellular events in which PI3-kinases are implicated, together with their conservation in evolution, indicates that they fulfil an essential role in cellular physiology.

Animals↗

PubMed1995

Casein kinase-1 phosphorylates the p75 tumor necrosis factor receptor and negatively regulates tumor necrosis factor signaling for apoptosis.

Cellular responses initiated by tumor necrosis factor (TNF) are mediated by two different cell surface receptors with respective molecular masses of 55 kDa (p55) and 75 kDa (p75). p55 is functional in almost every cell type and can independently transmit most biological activities of TNF. In contrast, TNF signaling via p75 seems so far largely restricted to cells of lymphoid origin, where it can induce proliferation, cytokine production, and/or apoptosis. The mechanisms that regulate TNF receptor activity are largely unknown. Here we report that the p75 of unstimulated p75-responsive PC60 T cells is phosphorylated on serine by a kinase activity present in p75 immune complexes. Several lines of evidence indicate that the latter kinase is casein kinase-1 (CK-1). Previous results have shown that the p75 TNF receptor is constitutively phosphorylated in vivo. Our data show that the latter in vivo phosphorylation is also at least partially due to CK-1. Pretreatment of cells with TNF had no detectable effect on p75 phosphorylation in vitro or in vivo. However, a specific CK-1 inhibitor potentiated TNF-induced apoptosis mediated by p75, suggesting an inhibitory role for phosphorylation by CK-1. Although in vivo p75 phosphorylation could be seen in both p75-unresponsive and p75-responsive cell lines, in vitro p75 phosphorylation in p75 coimmunoprecipitates could not be observed in cell lines that were biologically unresponsive to p75 stimulation. The latter observation further indicates a regulatory role for p75 phosphorylation in p75-mediated signaling. Taken together, our data demonstrate that the p75 TNF receptor is phosphorylated and associated with CK-1, which negatively regulates p75-mediated TNF signaling.

Apoptosis↗

PubMed1995

Generation and biological characterization of membrane-bound, uncleavable murine tumor necrosis factor.

Tumor necrosis factor (TNF) is produced as a membrane-bound, 26-kDa proform from which the mature, 17-kDa TNF subunit is released by proteolytic cleavage. In order to compare the biological activity of membrane-bound versus soluble TNF, mutational analysis of potential cleavage sites in murine TNF was carried out. The biological activity was assessed after transfection in L929 cells. Deletion of the first nine codons of the mature part of the murine TNF gene still led to the production of secretable TNF, indicating alternative cleavage sites separate from the -1/+1 junction. However, an additional deletion of 3 amino acids, generating TNF delta 1-12, resulted in a membrane-bound form of TNF. Site-directed mutagenesis revealed Lys11 as the critical residue for alternative cleavage. Mutation of this residue to Glu in a TNF delta 1-9 mutant gave rise to uncleavable, membrane-bound TNF with biological activities similar to wild-type TNF. Induction of apoptosis, proliferation, or cytokine production by triggering of either 55-kDa or 75-kDa TNF receptors in appropriate cell lines occurred efficiently both with soluble and with membrane-bound TNF. The latter was, however, less active in the cytotoxic assays on U937 cells in which the 75-kDa TNF receptor is not signaling, but contributes to maximal TNF activity by ligand passing. This indicates that membrane-bound TNF cannot be passed from the 75-kDa to the 55-kDa TNF receptor.

Amino Acid Sequence↗

PubMed1995

Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase.

Phosphoinositide-3 kinase activity is implicated in diverse cellular responses triggered by mammalian cell surface receptors and in the regulation of protein sorting in yeast. Receptors with intrinsic and associated tyrosine kinase activity recruit heterodimeric phosphoinositide-3 kinases that consist of p110 catalytic subunits and p85 adaptor molecules containing Src homology 2 (SH2) domains. A phosphoinositide-3 kinase isotype, p110 gamma, was cloned and characterized. The p110 gamma enzyme was activated in vitro by both the alpha and beta gamma subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) and did not interact with p85. A potential pleckstrin homology domain is located near its amino terminus. The p110 gamma isotype may link signaling through G protein-coupled receptors to the generation of phosphoinositide second messengers phosphorylated in the D-3 position.

Amino Acid Sequence↗

PubMed1995

A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p-Vps15p protein sorting system.

Phosphoinositide (PI) 3-kinases have been characterized as enzymes involved in receptor signal transduction in mammalian cells and in a complex which mediates protein trafficking in yeast. PI 3-kinases linked to receptors with intrinsic or associated tyrosine kinase activity are heterodimeric proteins, consisting of p85 adaptor and p110 catalytic subunits, which can generate the 3-phosphorylated forms of phosphatidylinositol (PtdIns), PtdIns4P and PtdIns(4,5)P2 as potential second messengers. Yeast Vps34p kinase, however, has a substrate specificity restricted to PtdIns and is a PtdIns 3-kinase. Here the molecular characterization of a new human PtdIns 3-kinase with extensive sequence homology to Vps34p is described. PtdIns 3-kinase does not associate with p85 and phosphorylates PtdIns, but not PtdIns4P or PtdIns(4,5)P2. In vivo PtdIns 3-kinase is in a complex with a cellular protein of 150 kDa, as detected by immunoprecipitation from human cells. Protein sequence analysis and cDNA cloning show that this 150 kDa protein is highly homologous to Vps15p, a 160 kDa protein serine/threonine kinase associated with yeast Vps34p. These results suggest that the major components of the yeast Vps intracellular trafficking complex are conserved in humans.

Amino Acid Sequence↗

PubMed1995

Both TNF receptors are required for TNF-mediated induction of apoptosis in PC60 cells.

A rat/mouse T cell hybridoma (PC60) was transfected either with human (h) TNF-R p55 (TNF-R55), p75 (TNF-R75) or both cDNAs. hTNF-R55 expression was below 50 molecules/cell, whereas the number of hTNF-R75 reached about 4000 molecules/cell. Only cells co-expressing the two types of receptor showed TNF-dependent apoptosis, in contrast to cells expressing similar levels of only one receptor type, indicating that both TNF-R55 and TNF-R75 are required. Stable co-transfection of the bcl-2 proto-oncogene largely prevented this TNF-mediated induction of apoptosis. We found that a high level of hTNF-R75 expression was essential for obtaining TNF-dependent apoptosis in PC60 cells in addition to a low number of hTNF-R55. Both receptors are signal transducing because simultaneous triggering of hTNF-R55 and hTNF-R75 by agonistic mAbs or by TNF-R-specific TNF muteins induced similar levels of apoptosis as wild-type hTNF. Apoptotic killing of only those lymphocytes expressing a high, induced level of TNF-R75, in addition to TNF-R55, may play a physiologically important role.

Animals↗

PubMed1995

Polar agents with differentiation inducing capacity potentiate tumor necrosis factor-mediated cytotoxicity in human myeloid cell lines.

Cotreatment or pretreatment of several human myeloid cell lines (KG1, HL60, U937, THP1) with the differentiation inducer DMSO was found to potentiate the antiproliferative and cytotoxic effects of TNF. In addition, TNF-resistant monocytic cell lines could be sensitized to TNF cytotoxicity by DMSO treatment. Other highly polar molecules, known to be potent differentiation inducers, showed similar effects to those of DMSO. The potentiating effect of DMSO was related neither to an up-regulation of TNF receptor expression nor to an alteration in the rate of TNF internalization and degradation. We present evidence that the TNF activities are p55 TNF receptor-mediated and are not due to insertion of TNF into lipid bilayers, an effect that could be susceptible to DMSO, as this component has been described to modify cell membrane characteristics. DMSO-induced potentiation of TNF cytostasis/cytotoxicity was restricted to myeloid leukemia cell lines. In non-myeloid cells such as fibrosarcomas, myosarcomas, thymomas, or carcinomas, DMSO was found either not to alter or to inhibit TNF-induced cell death. The latter results are in good agreement with data reported by others who suggested that DMSO could act as a scavenger of TNF-induced toxic radical formation. The potential correlation in myeloid cells between DMSO-induced changes in the cells' differentiation status and DMSO-enhanced TNF-susceptibility is discussed.

Animals↗

PubMed1994

Phosphatidylinositol-3-OH kinase as a direct target of Ras.

Ras (p21ras) interacts directly with the catalytic subunit of phosphatidylinositol-3-OH kinase in a GTP-dependent manner through the Ras effector site. In vivo, dominant negative Ras mutant N17 inhibits growth factor induced production of 3' phosphorylated phosphoinositides in PC12 cells, and transfection of Ras, but not Raf, into COS cells results in a large elevation in the level of these lipids. Therefore Ras can probably regulate phosphatidylinositol-3-OH kinase, providing a point of divergence in signalling pathways downstream of Ras.

Animals↗

PubMed1994

Functional requirement of the two TNF receptors for induction of apoptosis in PC60 cells and the role of mitochondria in TNF-induced cytotoxicity.

The rat/mouse T-cell hybridoma PC60 was transfected either with hTNF-R55 cDNA, hTNF-R75 cDNA, or both. Receptor-specific stimulation was achieved using agonistic monoclonal antibodies or receptor-specific muteins of hTNF. Either hTNF-R55 or hTNF-R75 could mediate the activation of NF-kappa B and the induction of GM-CSF, IL-6, and IFN-gamma. But only in cells carrying both hTNF-R55 and hTNF-R75, was TNF able to induce apoptosis. This apoptosis could be inhibited almost completely by cotransfection with human bcl-2 cDNA. Functional cooperation was observed between liganded and unliganded receptors for the induction of apoptosis. In vitro protein kinase activity was detected only in TNF-R75 immunoprecipitates from cells in which the receptor was signaling. Direct evidence was obtained for reactive oxygen intermediates of mitochondrial origin responsible for TNF-induced cytotoxicity in L929 cells.

Animals↗

PubMed1993

Sensitization of tumor cells to tumor necrosis factor action by the protein kinase inhibitor staurosporine.

Tumor necrosis factor (TNF), first described as a cytokine with tumor-necrotizing activity, is now known to be a pleiotropic molecule. The molecular mechanisms responsible for the cytotoxic activity of TNF on malignant cells are still largely unknown. In this study, we report that the protein kinase inhibitor staurosporine (56 to 1500 nM) increases about 500 times the in vitro cytotoxic activity of TNF for several murine and human tumor cell lines. Even some tumor cell lines which are resistant to TNF cytotoxicity could be sensitized to TNF killing by staurosporine. In the L929 fibrosarcoma cell line, staurosporine also enhanced the transcriptional activation of interleukin 6 synthesis by TNF (500-fold stimulation at 56 nM). At the biochemical level, staurosporine increased the TNF-mediated activation of phospholipases C and D and the transcription factor NF-kappa B in L929 cells. The TNF-sensitizing effect of staurosporine does not seem to be mediated by one of the currently known staurosporine-sensitive kinases, as various other inhibitors which also inhibit one or more of these kinases were not synergistic with TNF. Interestingly, staurosporine (1 microgram) also enhanced the in vivo antitumor activity of TNF against a murine tumor model (L929 fibrosarcoma) in athymic nude mice (Swiss-nu/nu; s.c. treatment). These results suggest that TNF responsiveness of tumor cells is regulated by a novel staurosporine-sensitive target and that the combination of TNF and staurosporine may open new strategies of tumor treatment.

Alkaloids↗

PubMed1993

Cell membrane permeabilization and cellular collapse, followed by loss of dehydrogenase activity: early events in tumour necrosis factor-induced cytotoxicity.

Early events in the cytotoxic response to tumour necrosis factor (TNF) of the murine fibrosarcoma cell lines L929 and WEHI164cl13 were assessed on a cell by cell basis using the fluorescent exclusion dye propidium iodide (PI) and analysis by flow cytometry. The rationale of this approach is based on the exclusion of PI by cells with intact membranes. PI-positive cells appeared within a few hours of TNF treatment and further accumulated with time at a TNF dose-dependent rate. Thus, TNF rapidly caused a breakdown of the barrier function of the membrane in these TNF-sensitive fibrosarcoma cell lines. On a time basis, membrane permeabilization was immediately followed by a sudden shrinkage of the cell and was accompanied by cell death, but preceded the inactivation of the mitochondrial succinate dehydrogenase by several hours. The latter enzymatic activity was measured by the MTT chromogenic assay. Cell death was determined on the basis of the capability of individual cells to produce a progeny in a clonogenicity assay. Both membrane permeabilization and cellular collapse were fast events that were completed within a very short time and may represent the direct cause for cell death. Opposed to this, loss of mitochondrial succinate dehydrogenase activity evolved more slowly, was initiated at a later time and apparently represents a post-lethal event, not directly linked to the TNF signal transduction pathway. Finally, the enhancing effect of the protein synthesis inhibitor cycloheximide on the various features of TNF-induced cytotoxicity was determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

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

Expression of an exogenous tumor necrosis factor (TNF) gene in TNF-sensitive cell lines confers resistance to TNF-mediated cell lysis.

TNF, a cytokine with cytotoxic activity on a variety of tumor cells, is mainly produced by macrophages; however, some tumor cell types of non-macrophage origin, apparently resistant to TNF-mediated cell lysis, can also produce TNF. It is not clear whether these cells were TNF-resistant a priori or whether protective mechanisms against toxicity of autocrine TNF may be induced in TNF-producing cells. Murine L929sA fibrosarcoma cells, which are highly sensitive to TNF cytotoxicity, were transfected with the neomycin resistance (neor) gene, alone or in combination with the human (h) or the murine (m) TNF gene. All exogenous genes were under control of the constitutive SV40 early promoter. After cotransfection, the number of neor colonies was 10 to 100% as compared with the number of colonies upon transfection with the neor gene alone. An appreciable fraction of these colonies (50-100%) constitutively produced biologically active TNF. mTNF-producing L929 cells were fully TNF resistant, whereas hTNF-producing cells showed partial TNF resistance. Specific TNF binding could not be detected on mTNF-producing L929sA transfectants, whereas hTNF-producing cells showed reduced TNF binding. Apparently, TNF gene expression, even in a priori TNF-sensitive cells, can induce mechanisms to prevent toxicity by both autocrine and exogenous TNF. No TNF resistance was induced by expression of a gene sequence encoding the 9-kDa membrane-bound presequence part of the 26-kDa mTNF proform. Expression of a mutant 26-kDa TNF gene coding for a quasi-inactive mature mTNF induced only weak TNF resistance as compared with the complete resistance obtained after transfection with the wild-type gene. These findings show that the membrane-bound TNF presequence as such is not sufficient for induction of TNF resistance and imply that the active site of mature TNF is involved in modulation of TNF responsiveness upon autocrine TNF production.

Animals↗

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

Plasma concentrations of pralidoxime methylsulphate in organophosphorus poisoned patients.

Using pharmacokinetic data from healthy human volunteers in a bicompartmental pharmacokinetic model, a repeated dose scheme for pralidoxime methylsulphate (Contrathion) was developed producing plasma levels remaining above the assumed "therapeutic concentration" of 4 mg.l-1. Using the same data, it was found that a concentration of 4 mg.l-1 could also be obtained by a loading dose of 4.42 mg.kg-1 followed by a maintenance dose of 2.14 mg.kg-1.h-1. In order to study the pharmacokinetic behaviour of pralidoxime in poisoned patients, this continuous infusion scheme was then applied in nine cases of organophosphorus poisoning (agents: ethyl parathion, ethyl and methyl parathion, dimethoate and bromophos), and the pralidoxime plasma levels were determined. The mean plasma levels obtained in the various patients varied between 2.12 and 9 mg.l-1. Pharmacokinetic data were calculated, giving a total body clearance of 0.57 +/- 0.27 l.kg-1.h-1 (mean +/- SD), an elimination half-life of 3.44 +/- 0.90 h, and a volume of distribution of 2.77 +/- 1.45 l.kg-1.

Dose-Response Relationship, Drug↗

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↗