Biomedical subjects

Patrick Legembre

Publications and source records attributed to Patrick Legembre.

11 recordsLinked to original sources

PubMed2007

Dominant-negative Fas mutation is reversed by down-expression of c-FLIP.

Fas triggering by agonistic antibodies or by its cognate ligand, FasL, induces apoptotic cell death, whereas mutation in the Fas death domain is associated with lymphoma progression. On prolonged culture in the presence of an agonistic anti-Fas antibody, we raised a Jurkat cell line resistant to agonistic antibodies but still sensitive to soluble FasL, which carried at the heterozygous state, a point mutation into the Fas death domain. Down-modulation of c-FLIP expression reversed the blockade of the Fas pathway. We show that the activation threshold for the Fas receptor is more easily overcome by multimeric FasL than by agonistic antibodies and that the increase of this threshold due to mutation in the Fas death domain can be overcome by acting on a downstream effector of the Fas signal, c-FLIP. These findings put forward a new approach to eradicate Fas-resistant tumor cells.

Amino Acid Sequence↗

PubMed2006

Modulation of Fas-mediated apoptosis by lipid rafts in T lymphocytes.

In type I cells, Fas-mediated cell death requires cytoplasmic membrane subdomains called microdomains or lipid rafts. On the contrary, Fas signaling is independent of these structures in type II cells. We report that in human T cells, CD28, CD59, and CD55 are all localized into lipid rafts and that CD28 is concentrated into microdomains enriched in ganglioside GM1, whereas CD59 and CD55 are not. Moreover, CD28 cross-linking leads to the formation of lipid raft clusters which exclude CD59 and CD55, and reciprocally. Coligation of Fas with CD55 or CD59 inhibits the apoptotic signal, whereas CD28 recruitment amplifies the Fas signaling pathway. Therefore, we conclude that 1) different types of microdomains exist on the cell surface, with distinct functional properties and 2) the recruitment of these distinct structures may differentially modulate the Fas pathway. Moreover, our results demonstrate that Fas-induced apoptosis can be controlled at the level of the cytoplasmic membrane.

Apoptosis↗

PubMed2005

Does CD95 have tumor promoting activities?

CD95 (APO-1/Fas) is an important inducer of the extrinsic apoptosis signaling pathway and therapy induced apoptosis of many tumor cells has been linked to the activity of CD95. Changes in the expression of CD95 and/or its ligand CD95L are frequently found in human cancer. The downregulation or mutation of CD95 has been proposed as a mechanism by which cancer cells avoid destruction by the immune system through reduced apoptosis sensitivity. CD95 has therefore been viewed as a tumor suppressor. Furthermore, increased CD95L concentration in tumor patients has been linked to tumor cells killing infiltrating lymphocytes in a process called "the tumor counter-attack". Recent data have illuminated unknown activities of CD95 in tumor cells with downregulated or mutated CD95 in the presence of increased CD95L. Under these conditions the stimulation of CD95 signals nonapoptotic pathways, activating NF-kappaB and MAP kinases for example, which may result in the induction of tumorigenic or prosurvival genes. A new model of CD95 functions is proposed in which CD95 is converted from a tumor suppressor to a tumor promotor by a single point mutation in one of the CD95 alleles, a situation frequently found in advanced human cancer, resulting in apoptosis resistance and activation of tumorigenic pathways.

Animals↗

PubMed2005

Amplification of Fas-mediated apoptosis in type II cells via microdomain recruitment.

Fas triggers apoptosis via the caspase cascade when bound to its ligand FasL. In type I cells, Fas is concentrated into the plasma membrane lipid rafts, and these domains are required for the apoptotic signal to occur. In contrast, Fas is excluded from the microdomains in type II cells. We report that the coligation with Fas of the membrane receptor CD28 strongly increases Fas-induced apoptosis in type II T lymphocytes, whereas it has no effect in a type I cell line. The effect of CD28 is independent of its intracellular region and requires the recruitment of the microdomains. Indeed, upon CD28 costimulation, Fas is redistributed in the lipid rafts, and their disruption with a cholesterol chelator abrogates the effect of CD28. The microdomain-mediated cell death amplification does not alter death-induced signaling complex formation and is mediated by the enhancement of the mitochondrial apoptotic pathway. These findings indicate that the sensitivity to Fas-induced apoptosis of type II cells can be amplified in vivo by the recruitment of lipid rafts following interactions between nonapoptotic ligand/receptor pairs during cell-to-cell contacts.

Animals↗

PubMed2004

The relevance of NF-kappaB for CD95 signaling in tumor cells.

Most members of the death receptor family including CD95 (APO-1/Fas) have been shown to induce both apoptosis as well as non-apoptotic pathways depending on the tissue and the circumstances. One of the non-apoptotic pathways emanating from CD95, activation of NF-kappaB, has recently been demonstrated to regulate invasiveness of apoptosis resistant tumor cells. In contrast, activation of NF-kappaB in apoptosing cells is believed to be suppressed due to cleavage of various NF-kappaB pathway components by active caspases that execute apoptosis. We now present data demonstrating that in certain highly CD95 apoptosis sensitive cells NF-kappaB is robustly activated. In fact overexpression of apoptosis inhibitors such as Bcl-2 or c-FLIPL in these cells results in decreased activation of NF-kappaB through CD95. We propose a model in which NF-kappaB is generally activated in certain cells but may have different functions depending on whether cells are programmed to die or to survive.

CASP8 and FADD-Like Apoptosis Regulating Protein↗

PubMed2004

Identification of SNF1/AMP kinase-related kinase as an NF-kappaB-regulated anti-apoptotic kinase involved in CD95-induced motility and invasiveness.

The death receptor CD95 (APO-1/Fas) induces apoptosis in many tissues. However, in apoptosis-resistant tumor cells, stimulation of CD95 induces up-regulation of a defined number of mostly anti-apoptotic genes, resulting in increased motility and invasiveness of tumor cells. The majority of these genes are known NF-kappaB target genes. We have identified one of the CD95-regulated genes as the serine/threonine kinase (SNF1/AMP kinase-related kinase (SNARK)), which is induced in response to various forms of metabolic stress. We demonstrate that up-regulation of SNARK in response to CD95 ligand and tumor necrosis factor alpha depends on activation of NF-kappaB. Overexpression of SNARK rendered tumor cells more resistant, whereas a kinase-inactive mutant of SNARK sensitized cells to CD95-mediated apoptosis. Furthermore, small interfering RNA-mediated knockdown of SNARK increased the sensitivity of tumor cells to CD95 ligand- and TRAIL-induced apoptosis. Importantly, cells with reduced expression of SNARK also showed reduced motility and invasiveness in response to CD95 engagement. SNARK therefore represents an NF-kappaB-regulated anti-apoptotic gene that contributes to the tumor-promoting activity of CD95 in apoptosis-resistant tumor cells.

Apoptosis↗

PubMed2004

CD95 ligand induces motility and invasiveness of apoptosis-resistant tumor cells.

The apoptosis-inducing death receptor CD95 (APO-1/Fas) controls the homeostasis of many tissues. Despite its apoptotic potential, most human tumors are refractory to the cytotoxic effects of CD95 ligand. We now show that CD95 stimulation of multiple apoptosis-resistant tumor cells by CD95 ligand induces increased motility and invasiveness, a response much less efficiently triggered by TNFalpha or TRAIL. Three signaling pathways resulting in activation of NF-kappaB, Erk1/2 and caspase-8 were found to be important to this novel activity of CD95. Gene chip analyses of a CD95-stimulated tumor cell line identified a number of potential survival genes and genes that are known to regulate increased motility and invasiveness of tumor cells to be induced. Among these genes, urokinase plasminogen activator was found to be required for the CD95 ligand-induced motility and invasiveness. Our data suggest that CD95L, which is found elevated in many human cancer patients, has tumorigenic activities on human cancer cells. This could become highly relevant during chemotherapy, which can cause upregulation of CD95 ligand by both tumor and nontumor cells.

Apoptosis↗

PubMed2004

Induction of apoptosis and activation of NF-kappaB by CD95 require different signalling thresholds.

Mutations in the death domain of the death receptor CD95 (APO-1/Fas) cause lymphoproliferation and autoimmune disease in both lpr(cg) mice and in patients with autoimmune lymphoproliferative syndrome (ALPS) type Ia. By testing lymphocytes from ALPS type Ia patients, comparing heterozygous with homozygous lpr(cg) mice and coexpressing wild-type and mutant CD95 receptors, we demonstrate that induction of apoptosis requires two wild-type alleles of CD95. By contrast, nuclear factor-kappaB (NF-kappaB) can be fully activated in cells expressing both a mutant and a wild-type CD95 allele, suggesting different thresholds to activate the two signalling pathways. This was confirmed by testing lymphocytes from heterozygous lpr mice, which showed reduced sensitivity to CD95-mediated apoptosis but normal activation of NF-kappaB when compared with wild-type mice. Mutations in CD95 may eliminate the tumour-suppressive function of CD95, at the same time allowing induction of survival or proliferative pathways, which could contribute to the increased risk for lymphoma seen in ALPS type Ia patients.

Alleles↗

PubMed2003

Cutting edge: SDS-stable Fas microaggregates: an early event of Fas activation occurring with agonistic anti-Fas antibody but not with Fas ligand.

The 45 kDa Fas or CD95 receptor triggers apoptosis via the caspase cascade when stimulated by its ligand FasL or by agonistic Abs. Activated Fas receptors seem to oligomerize very early into SDS-stable and reducing agent-resistant microaggregates of 200-250 kDa on SDS-PAGE. However, these microaggregates have so far only been reported using agonistic anti-Fas Abs, and no results have been reported using FasL. Here, we demonstrate that the microaggregates do not form in response to FasL, while they always appear in response to the agonistic Ab, in four different cell lines and in normal lymphocytes from human blood. Therefore, the Fas microaggregates are not required for the induction of apoptosis via FasL. These results also suggest that subtle differences exist in the apoptotic pathways triggered by anti-Fas agonistic Abs and by FasL.

Antibodies, Monoclonal↗

PubMed2003

Two CD95 tumor classes with different sensitivities to antitumor drugs.

CD95 type I and II cells differ in their dependence on mitochondria to execute apoptosis, because antiapoptotic members of the Bcl-2 family render only type II cells resistant to death receptor-induced apoptosis. They can also be distinguished by a more efficient formation of the death-inducing signaling complex in type I cells. We have identified a soluble form of CD95 ligand (S2) that is cytotoxic to type II cells but does not kill type I cells. By testing 58 tumor cell lines of the National Cancer Institute's anticancer drug-screening panel for apoptosis sensitivity to S2 and performing death-inducing signaling complex analyses, we determined that half of the CD95-sensitive cells are type I and half are type II. Most of the type I cell lines fall into a distinct class of tumor cells expressing mesenchymal-like genes, whereas the type II cell lines preferentially express epithelium-like markers. This suggests that type I and II tumor cells represent different stages of carcinogenesis that resemble the epithelial-mesenchymal transition. We then screened the National Cancer Institute database of >42,000 compounds for reagents with patterns of growth inhibition that correlated with either type I or type II cell lines and found that actin-binding compounds selectively inhibited growth of type I cells, whereas tubulin-interacting compounds inhibited growth of type II cells. Our analysis reveals fundamental differences in programs of gene expression between type I and type II cells and could impact the way actin- and microtubule-disrupting antitumor agents are used in tumor therapy.

Antineoplastic Agents↗

PubMed2003

Flt3-ligand induces adhesion of haematopoietic progenitor cells via a very late antigen (VLA)-4- and VLA-5-dependent mechanism.

The adhesion of haematopoietic progenitor cells (HPC) to the bone marrow microenvironment is a process regulated by cytokines. In this study, we have shown that flt3-ligand (FL), a growth factor that controls early haematopoiesis, regulated the function and expression of the beta-1 integrins, very late antigen (VLA)-4 and VLA-5 on HPC. The modulation of the adhesiveness of HPC by FL was studied by adhesion assays on umbilical vein endothelial cells (HUVEC). Stimulation by FL induced two peaks of increased adhesiveness of HPC. The first peak was at around 30 min and was mechanistically related to an activation of the beta-1 integrins, mainly VLA-4 and VLA-5. The second peak was at around 12 h and was related to increased expression of VLA-4 and VLA-5. The control of HPC adhesiveness by FL is a previously unreported property of FL that may be important for the homing and the retention of flt3-expressing HPC within the bone marrow microenvironment.

Antigens, CD34↗