Biomedical subjects

D Vercammen

Publications and source records attributed to D Vercammen.

16 recordsLinked to original sources

PubMed2001

Apoptotic and necrotic cell death induced by death domain receptors.

Apoptosis and necrosis are two distinct forms of cell death. Caspases are indispensable as initiators and effectors of apoptotic cell death and are involved in many of the morphological and biochemical features of apoptosis. Major changes in mitochondrial membrane integrity and release of proapoptotic factors, such as cytochrome c from the mitochondrial intermembrane space, play an important sensor and amplifying role during apoptotic cell death. In vitro studies of cell death in cell lines have revealed that inhibition of the classical caspase-dependent apoptotic pathway leads in several cases to necrotic cell death. Thus, the same cell death stimulus can result either in apoptotic or necrotic cell death, depending on the availability of activated caspase. Therefore, death domain receptors may initiate an active caspase-independent necrotic signaling pathway. In this review, we describe what is known about the apoptotic and necrotic cell death pathways. Principal elements of necrosis include mitochondrial oxidative phosphorylation, reactive oxygen production, and non-caspase proteolytic cascades depending on serine proteases, calpains, or cathepsins.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Death receptor-induced apoptotic and necrotic cell death: differential role of caspases and mitochondria.

In L929sAhFas cells, tumor necrosis factor (TNF) leads to necrotic cell death, whereas agonistic anti-Fas antibodies elicit apoptotic cell death. Apoptosis, but not necrosis, is correlated with a rapid externalization of phosphatidylserine and the appearance of a hypoploid population. During necrosis no cytosolic and organelle-associated active caspase-3 and -7 fragments are detectable. The necrotic process does not involve proteolytic generation of truncated Bid; moreover, no mitochondrial release of cytochrome c is observed. Bcl-2 overexpression slows down the onset of necrotic cell death. In the case of apoptosis, active caspases are released to the culture supernatant, coinciding with the release of lactate dehydrogenase. Following necrosis, mainly unprocessed forms of caspases are released. Both TNF-induced necrosis and necrosis induced by anti-Fas in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone are prevented by the serine protease inhibitor N-tosyl-L-phenylalanine chloromethylketone and the oxygen radical scavenger butylated hydroxyanisole, while Fas-induced apoptosis is not affected.

Animals↗

PubMed2000

Structure/Function analysis of p55 tumor necrosis factor receptor and fas-associated death domain. Effect on necrosis in L929sA cells.

Tumor necrosis factor (TNF) induces a typical apoptotic cell death program in various cell lines by interacting with the p55 tumor necrosis factor receptor (TNF-R55). In contrast, triggering of the fibrosarcoma cell line L929sA gives rise to characteristic cellular changes resulting in necrosis. The intracellular domain of TNF-R55 can be subdivided into two parts: a membrane-proximal domain (amino acids 202-325) and a C-terminal death domain (DD) (amino acids 326-413), which has been shown to be necessary and sufficient for apoptosis. Structure/function analysis of TNF-R55-mediated necrosis in L929sA cells demonstrated that initiation of necrotic cell death, as defined by swelling of the cells, rapid membrane permeabilization, absence of nuclear condensation, absence of DNA hypoploidy, and generation of mitochondrial reactive oxygen intermediates, is also confined to the DD. The striking synergistic effect of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone on TNF-induced necrosis was also observed with receptors solely containing the DD. TNF-R55-mediated necrosis is not affected by the dominant negative deletion mutant of the Fas-associated death domain (FADD-(80-205)) that lacks the N-terminal death effector domain. Moreover, overexpression of FADD-(80-205) in L929sA is cytotoxic and insensitive to CrmA, while the cytotoxicity due to overexpression of the deletion mutant FADD-(1-111) lacking the DD is prevented by CrmA. These results demonstrate that the death domain of FADD can elicit an active necrotic cell death pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Phosphatidyl serine exposure during apoptosis precedes release of cytochrome c and decrease in mitochondrial transmembrane potential.

Time kinetics of phosphatidyl serine (PS) exposure were compared to other apoptotic parameters following different apoptotic stimuli. Our data indicate that anti-Fas treatment of L929sAhFas cells results in rapid exposure of PS, which precedes decrease in mitochondrial transmembrane potential (DeltaPsi(m)) and release of cytochrome c, indicating that PS exposure occurs independently of these mitochondrial events. Also during TNF-, etoposide- or staurosporine-mediated apoptosis in PC60 RI/RII cells, PS-positive cells were observed before they had a decreased DeltaPsi(m). However, during growth factor depletion-induced death of 32D cells, both phenomena seemed to occur at the same time.

Animals↗

PubMed2000

A role for potassium in TNF-induced apoptosis and gene-induction in human and rodent tumour cell lines.

Rat/mouse T cell hybridoma-derived PC60 R55/R75 cells were used as a model to study the role of intracellular potassium in TNF-induced apoptosis and gene induction. A reduction of intracellular potassium with nigericin or valinomycin (ionophores), or ouabain (Na(+)/K(+)-ATPase inhibitor) sensitized PC60 R55/R75 cells to TNF-induced apoptosis. TNF-induced GM-CSF release in PC60 R55/R75 cells was enhanced by nigericin or ouabain. Similar results were obtained with human cervix carcinoma cells HeLaH21 exposed to TNF. These results suggest a role for intracellular potassium in TNF-induced apoptosis and gene induction.

Animals↗

PubMed1999

Redox regulation of TNF signaling.

TNF is produced during inflammation and induces, among other activities, cell death in sensitive tumour cells. We previously reported an increased generation of ROS in TNF-treated L929 fibrosarcoma cells prior to cell death. These ROS are of mitochondrial origin and participate in the cell death process. Presently, we focus on the identification of parameters that control ROS production and subsequent cytotoxicity. From the cytotoxic properties and susceptibility to scavenging of TNF-induced ROS as compared to pro-oxidant-induced ROS we conclude that TNF-mediated ROS generation and their lethal action are confined to the inner mitochondrial membrane. Oxidative substrates, electron-transport inhibitors, glutathione and thiol-reactive agents but also caspase inhibitors modulate TNF-induced ROS production and imply the existence of a negative regulator of ROS production. Inactivation of this regulator by a TNF-induced reduction of NAD(P)H levels and/or formation of intraprotein disulfides would be responsible for ROS generation.

Animals↗

PubMed1998

Differential activation of phospholipases during necrosis or apoptosis: a comparative study using tumor necrosis factor and anti-Fas antibodies.

Phospholipases generate important secondary messengers in several cellular processes, including cell death. Tumor necrosis factor (TNF) can induce two distinct modes of cell death, viz. necrosis and apoptosis. Here we demonstrate that phospholipase D (PLD) and cytosolic phospholipase A2 (cPLA2) are differentially activated during TNF-induced necrosis or apoptosis. Moreover, a comparative study using TNF and anti-Fas antibodies as cell death stimuli showed that PLD and cPLA2 are specifically activated by TNF. These results indicate that both the mode of cell death and the type of death stimulus determine the potential role of phospholipases as generators of secondary messengers.

Animals↗

PubMed1998

Dual signaling of the Fas receptor: initiation of both apoptotic and necrotic cell death pathways.

Murine L929 fibrosarcoma cells were transfected with the human Fas (APO-1/CD95) receptor, and the role of various caspases in Fas-mediated cell death was assessed. Proteolytic activation of procaspase-3 and -7 was shown by Western analysis. Acetyl-Tyr-Val-Ala-Asp-chloromethylketone and benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone++ +, tetrapeptide inhibitors of caspase-1- and caspase-3-like proteases, respectively, failed to block Fas-induced apoptosis. Unexpectedly, the broad-spectrum caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone and benzyloxycarbonyl-Asp(OMe)-fluoromethylketone rendered the cells even more sensitive to Fas-mediated cell death, as measured after 18 h incubation. However, when the process was followed microscopically, it became clear that anti-Fas-induced apoptosis of Fas-transfected L929 cells was blocked during the first 3 h, and subsequently the cells died by necrosis. As in tumor necrosis factor (TNF)-induced necrosis, Fas treatment led to accumulation of reactive oxygen radicals, and Fas-mediated necrosis was inhibited by the oxygen radical scavenger butylated hydroxyanisole. However, in contrast to TNF, anti-Fas did not activate the nuclear factor kappaB under these necrotic conditions. These results demonstrate the existence of two different pathways originating from the Fas receptor, one rapidly leading to apoptosis, and, if this apoptotic pathway is blocked by caspase inhibitors, a second directing the cells to necrosis and involving oxygen radical production.

Amino Acid Chloromethyl Ketones↗

PubMed1998

Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor.

Murine L929 fibrosarcoma cells treated with tumor necrosis factor (TNF) rapidly die in a necrotic way, due to excessive formation of reactive oxygen intermediates. We investigated the role of caspases in the necrotic cell death pathway. When the cytokine response modifier A (CrmA), a serpin-like caspase inhibitor of viral origin, was stably overexpressed in L929 cells, the latter became 1,000-fold more sensitive to TNF-mediated cell death. In addition, TNF sensitization was also observed when the cells were pretreated with Ac-YVAD-cmk or zDEVD-fmk, which inhibits caspase-1- and caspase-3-like proteases, respectively. zVAD-fmk and zD-fmk, two broad-spectrum inhibitors of caspases, also rendered the cells more sensitive, since the half-maximal dose for TNF-mediated necrosis decreased by a factor of 1,000. The presence of zVAD-fmk also resulted in a more rapid increase of TNF-mediated production of oxygen radicals. zVAD-fmk-dependent sensitization of TNF cytotoxicity could be completely inhibited by the oxygen radical scavenger butylated hydroxyanisole. These results indicate an involvement of caspases in protection against TNF-induced formation of oxygen radicals and necrosis.

Amino Acid Chloromethyl Ketones↗

PubMed1998

The 55-kDa tumor necrosis factor receptor induces clustering of mitochondria through its membrane-proximal region.

The cytokine tumor necrosis factor (TNF) activates diverse signaling molecules resulting in gene expression, differentiation, and/or cell death. Here we report a novel feature induced by TNF, namely translocation of mitochondria from a dispersed distribution to a perinuclear cluster. Mitochondrial translocation correlated with sensitivity to the cell death-inducing activity of TNF and was mediated by the 55-kDa TNF receptor (TNF-R55), but not by Fas, indicating that the signaling pathway requires a TNF-R55-specific but death domain-independent signal. Indeed, using L929 cells that express mutant TNF-R55, we showed that the membrane-proximal region of TNF-R55 was essential for signaling to mitochondrial translocation. In the absence of translocation, the cell death response was markedly delayed, pointing to a cooperative effect on cell death. Translocation of mitochondria, although dependent on the microtubules, was not imposed by the latter and was equally induced by TNF-independent immunoinhibition of the motor protein kinesin. Additionally, immunoinhibition with antibody directed against the tail domain of kinesin synergized with TNF-induced cell death. Based on this functional mimicry, we propose that a TNF-R55 membrane-proximal region-dependent signal impedes mitochondria-associated kinesin, resulting in cooperation with the TNF-R55 death domain-induced cytotoxic response and causing the observed clustering of mitochondria.

Animals↗

PubMed1998

Sensitization of tnf-induced apoptosis with polyamine synthesis inhibitors in different human and murine tumour cell lines.

Rat/mouse T cell hybridoma-derived PC60 R55/R75 cells were used as a model to study tumour necrosis factor (TNF)-induced apoptosis. The role of ornithine decarboxylase (ODC) activity and polyamines in this process was investigated. In PC60 R55/R75 cells, TNF-induced ODC activity was completely suppressed by externally added spermine (Spm). TNF decreased the intracellular levels of the three polyamines Spm, spermidine (Spd) and putrescine (Put). A reduction of the intracellular [Spm] with methylglyoxal bis(quanyl hydrasone) (MGBG), CGP48644a, or bis(ethyl)norspermine (BENSpm), clearly sensitized the cells towards the apoptotic effect of TNF. Conversely, an increase in intracellular [Spm] with DFMO or externally added Spm reduced cellular sensitivity. Similar results were obtained after TNF treatment of the human cell lines Kym 39A6 (rhabdomyosarcoma), HeLaH21 (cervix carcinoma) and U937 (histocytoma) and after alphaFas treatment of HeLaH21, U937 and CEM-CM3 (human T cell line). These results suggest that a decrease of intracellular Spm levels rather then ODC activity per se is involved in the sensitization towards apoptosis induced by TNF or alphaFas.

Amidines↗

PubMed1997

Tumour necrosis factor-induced necrosis versus anti-Fas-induced apoptosis in L929 cells.

Murine fibrosarcoma L929 cells were transfected with human Fas cDNA. The mode of cell death was analysed following treatment either with tumour necrosis factor (TNF) or with agonistic antibodies to Fas. While triggering of the TNF receptors led to necrosis, clustering of the Fas antigen resulted in apoptotic cell death. N-tosyl-l-phenylalanine chloromethyl ketone and Nalpha-p-tosyl-l-lysine chloromethyl ketone, two serine protease inhibitors, has a protective effect on TNF-induced killing, while Fas-mediated cell death was rather enhanced. Lithium chloride, which had a synergistic effect on TNF cytotoxicity, did not affect Fas-mediated death, whereas staurosporine had an enhancing effect on both types of cell death. Aphidicolin and hydroxyurea, inhibitors of DNA synthesis, were able to sensitize cells to Fas-induced killing, but had no effect on TNF cytotoxicity. Finally, we demonstrate that the effect of increasing concentrations of actinomycin D or cycloheximide is very different for the two types of cell killing. We conclude that either necrosis or apoptosis can occur in the same cell type, depending on the trigger, and that, although both pathways perhaps may share some cellular components, signal transduction is different for the two types of cell death.

Adjuvants, Immunologic↗

PubMed1995

Cytotoxicity in L929 murine fibrosarcoma cells after triggering of transfected human p75 tumour necrosis factor (TNF) receptor is mediated by endogenous murine TNF.

We compared the biological function of the human tumor necrosis factor receptors p55 (hTNF-R55) and p75 (hTNF-R75) expressed in the murine (m) fibrosarcoma cell line L929. Receptor-specific triggering of hTNF-R55 in transfected L929 cells by agonistic monoclonal antibodies or hTNF-R32WS86T, a hTNF-R55-specific mutant of hTNF, resulted in cytotoxicity. Specific clustering of hTNF-R75 in transfected L929 cells by agonistic monoclonal antibodies or hTNF-D143F, a hTNF-R75-specific mutant of hTNF also induced cytotoxicity, albeit at low level. In both cases, the cytotoxic activity of receptor clustering could be synergized by addition of 20 mM LiCl. Remarkably, cytotoxicity induced after R75 triggering in transfected L929 cells could be completely abolished by addition of neutralizing anti-mTNF antibodies, in contrast to cell killing seen after specific R55 clustering. No soluble mTNF could be demonstrated using a sensitive biological assay, although L929 cells were expressing low levels of mTNF-specific mRNA as shown by PCR. These data clearly demonstrate that minute amounts of endogenously produced TNF can be a key mediator in R75-mediated cytotoxicity. Presumably, the latter efficiently traps the ligand and transfers it to TNF-R55, and/or by binding it, protects the endogenously made TNF from inactivation.

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↗

PubMed1992

Functional characterization of the human tumor necrosis factor receptor p75 in a transfected rat/mouse T cell hybridoma.

We investigated the biological role of the human tumor necrosis factor p75 (hTNF-R75), making use of the species specificity of TNF responses in murine (m) T cell lines. Several TNF-mediated activities on mouse T cells, such as cytokine induction or proliferation, showed a 100-500-fold difference in specific biological activity between mTNF and hTNF. After transfection of hTNF-R75 cDNA in a rat/mouse T cell hybridoma (PC60), however, the 100-fold lower specific biological activity of hTNF was converted to the same specific biological activity as mTNF. The TNF-mediated induction of granulocyte/macrophage colony-stimulating factor was strongly synergized by the addition of interleukin 1. In the presence of the latter cytokine, ligand-competing monoclonal antibodies against hTNF-R75 (utr-1, utr-2, utr-3) were agonistic on transfected PC60 cells. This agonistic activity was further enhanced by crosslinking with sheep anti-murine immunoglobulin antibodies. These data provide direct evidence for a functional role of TNF-R75, without ligand-dependent TNF-R55 involvement, in the induction of cytokine secretion in T cells.

Animals↗

PubMedDate not supplied

TNF-induced intracellular signaling leading to gene induction or to cytotoxicity by necrosis or by apoptosis.

TNF-induced apoptosis, e.g. in murine PC60 cells, requires the TNF receptor p55 (TNF-R55) and the TNF receptor p75 (TNF-R75); the latter even does not have to be triggered. The intracellular domain of TNF-R55 can be activated in the cytosol by linking it to the trimeric CAT protein; induction of this fusion protein leads to a full TNF response. A new MAP kinase, p38, has been shown to be also activated by TNF. This activation is essential for gene induction, but not for cytotoxicity in L929 cells. TNF treatment of L929 leads to reactive oxygen formation in the mitochondria, resulting in cell death by necrosis. TNF treatment of many other cell types results in apoptosis, and this process involves activation of one or more ICE homologs (IHO). In the mouse, seven cysteine proteases of the IHO family have been cloned and partially characterized. One or more of these IHOs is involved in cell killing by proteolysis of critical substrate(s). One substrate, which may be a key effector molecule in the apoptotic process, is PITSLRE kinase.

Animals↗