Molecular mechanisms and effects of a rasgap-derived peptide on metastatic progression

Cancer is the second leading cause of mortality worldwide. Cancer progression leads to metastasis formation, which accounts for more than ninety percent of cancer-related death. Metastases are more difficult to be surgically removed because of their invasive behavior and shape. In addition, during their transformation journey, they become more and more resistant to anticancer drugs. Significant improvements have been achieved in therapy against cancer in recent years but targeting the metastatic cascade remains the Achilles heel of the cure against cancer. A First step in the metastatic process is the escape of cancer cells from the primary tumor site. This involves an increase in cell motility and the concomitant ability to clear a path through the extracellular matrix. From a therapeutic point of view, inhibition of cell migration is a logical approach to develop anti-metastatic drugs. Our lab previously developed a cell permeable peptide derived from a caspase-3-generaied fragment of the RasGAP protein called TAT-RasGAP317-326. This peptide efficiently and specifically sensitizes cancer cells to chemotherapy- and radiotherapy-induced ceil death, which allows decreasing the anticancer drug doses and eventually their associated side- effects. In the present study we discovered that TAT-RasGAP317.326 also increases cell adhesion which was associated with inhibition of cell migration and invasion into the extracellular matrix. The ability of TAT-RasGAP317.326 to increase ceil adhesion involves the dramatic depolymerization of actin cytoskekton together with redistribution of focal adhesions. We found that the inhibitory effects on migration were mediated by a RhoGAP tumor and metastasis suppressor cailed DLC1 (Deleted in Liver Cancer 1). Moreover. DEC 1 was found to be a direct RasGAP-interacting protein and this interaction requires the RasGAP tryptophan 317 residue, the very first RasGAP residue of TAT-RasGAP317.326. We then evaluated the roie of RasGAP fragments in the in vivo metastatic cascade. We found that breast cancer cells overexpressing the parental RasGAP fragment, to which the TAT-RasGAP317.326 peptide belongs, have a markedly decreased ability to form lung metastases. Unfortunately, we were not able to recapitulate these an ti-metastatic effects when TAT-RasGAP317.326 was injected. However, we later understood that this was due to the fact that TAT-RasGAP317.326 was not properly delivered to the primary tumors. Further work, aimed at better understanding of how TAT-RasGAP317.326 functions, revealed that the ten amino acid TAT-RasGAP317.326 peptide could, be narrowed down to a three amino acid TAT-RasGAP317.329 peptide while keeping its sensitizer activity. In parallel, investigations on the RasGAP-DLCl binding indicated that the arginine linger of the DLC1 GAP domain is required for this interaction, which suggests that TAT-RasGAP317.326 modulates the GAP activity of DLC1. Additional work should be performed to fully elucidate its mechanism of action and render TAT-RasGAP317.326 usable as a tool to fight cancer on two fronts, by improving chemotherapy and preventing metastatic progression. - Le cancer est la deuxième cause de mortalité dans le monde. La formation de métastases est la dernière étape de la progression cancéreuse et représente plus du nonante pour cent des morts induites par le cancer. De par leur morphologie et comportement invasifs, ii est difficile d'avoir recours à la chirurgie pour exciser des métastases. De plus, les cellules cancéreuses en progression deviennent souvent de plus en plus résistantes aux drogues anticancéreuses. Ces dernières années, des avancements significatifs ont contribué à l'amélioration de la lutte contre le cancer. Néanmoins, pouvoir cibler spécifiquement la cascade métastatique demeure cependant le talon d'Achille des thérapies anticancéreuses. Une première étape dans ie processus métastatique est l'évasion des cellules cancéreuses du site de la tumeur primaire. Ceci requiert une augmentation de la motiliié cellulaire couplée à la capacité de se frayer un chemin au sein de la matrice extracelluiaire. D'un point de vue thérapeutique, inhiber la migration cellulaire est une approche attrayante. Notre laboratoire a développé un peptide, nommé TAT-RasGAP317.326 dérivé d'un fragment qui est lui-même le résultat du clivage de la protéine RasGAP par la caspase-3. Ce peptide est capable de pénétrer les cellules cancéreuses et de les sensibiliser spécifiquement à la mort induite par la radiothérapie et la chimiothérapie. La finalité des effets de ce peptide est de pouvoir diminuer les doses des traitements anti-cancéreux et donc des effets secondaires qu'ils engendrent. Dans cette étude, nous avons découvert que TAT-RasGAP317.326 augmente l'adhésion des cellules et inhibe la migration cellulaire ainsi que l'invasion des cellules à travers une matrice extracellulaire. La capacité de TAT-RasGAP317.326 à induire l'adhésion repose sur ia dépolymérisation du cytosquelette d'actine associée à une redistribution des points d'ancrage cellulaire. Nous avons découvert que l'inhibition de ia migration par TAT-RasGAP317.326 nécessitait la présence d'un suppresseur de tumeur et de métastases appelé DLC1 (Deleted in Liver Cancer l), qui par ailleurs s'avère aussi être une protéine RhoGAP. De plus, nous avons aussi trouvé que DLC1 était un partenaire d'interaction de RasGAP et que cette interaction s'effectuait via l'acide aminé tryptophane 317 de RasGAP. qui s'avère être le premier acide aminé du peptide TAT-RasGAP317.326. Nous avons ensuite évalué le rôle joué par certains fragments de RasGAP dans le processus de métastatisation. Dans ce contexte, des cellules de cancer du sein qui sur-expriment un fragment de RasGAP contenant la séquence TAT-RasGAP317.326 ont vu leur potentiel métastatique diminuer drastiquerment. Malheureusement, aucun effet anti-métastatique n'a été obtenu après injection de TAT-RasGAP317.326 dans les souris. Cependant, nous avons réalisé rétrospectivement que TAT-RasGAP317.326 n'était pas correctement délivré à la tumeur primaire, ce qui nous empêche de tirer des conclusions sur le rôle anti-métastatique de ce peptide. La suite de cette étude visant à mieux comprendre comment TAT-RasGAP317.326 agit, a mené à la découverte que les dix acides aminés de TAT-RasGAP317.326 pouvaient être réduits à trois acides aminés, TAT-RasGAP317.329, tout en gardant l'effet sensibilisateur à la chimiothérapie. En visant à élucider le mode d'interaction entre RasGAP et DLC1, nous avons découvert qu'un acide aminé nécessaire à l'activité GAP de DLC1 était requis pour lier RasGAP, ce qui laisse présager que TAT-RasGAp317.32c, module i'activité GAP de DLC1. Des travaux supplémentaires doivent encore être effectués pour complètement élucider les mécanismes d'action de TAT-RasGAP317.326 et afin de pouvoir l'utiliser comme un outil pour combattre le cancer sur deux fronts, en améliorant les chimiothérapies et en inhibant la formation de métastases.

Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex.

The membrane-bound form of Fas ligand (FasL) signals apoptosis in target cells through engagement of the death receptor Fas, whereas the proteolytically processed, soluble form of FasL does not induce cell death. However, soluble FasL can be rendered active upon cross-linking. Since the minimal extent of oligomerization of FasL that exerts cytotoxicity is unknown, we engineered hexameric proteins containing two trimers of FasL within the same molecule. This was achieved by fusing FasL to the Fc portion of immunoglobulin G1 or to the collagen domain of ACRP30/adiponectin. Trimeric FasL and hexameric FasL both bound to Fas, but only the hexameric forms were highly cytotoxic and competent to signal apoptosis via formation of a death-inducing signaling complex. Three sequential early events in Fas-mediated apoptosis could be dissected, namely, receptor binding, receptor activation, and recruitment of intracellular signaling molecules, each of which occurred independently of the subsequent one. These results demonstrate that the limited oligomerization of FasL, and most likely of some other tumor necrosis factor family ligands such as CD40L, is required for triggering of the signaling pathways.

Brain damage in methylmalonic aciduria: 2-methylcitrate induces cerebral ammonium accumulation and apoptosis in 3D organotypic brain cell cultures.

BACKGROUND: Methylmalonic aciduria is an inborn error of metabolism characterized by accumulation of methylmalonate (MMA), propionate and 2-methylcitrate (2-MCA) in body fluids. Early diagnosis and current treatment strategies aimed at limiting the production of these metabolites are only partially effective in preventing neurological damage. METHODS: To explore the metabolic consequences of methylmalonic aciduria on the brain, we used 3D organotypic brain cell cultures from rat embryos. We challenged the cultures at two different developmental stages with 1 mM MMA, propionate or 2-MCA applied 6 times every 12 h. In a dose-response experiment cultures were challenged with 0.01, 0.1, 0.33 and 1 mM 2-MCA. Immunohistochemical staining for different brain cell markers were used to assess cell viability, morphology and differentiation. Significant changes were validated by western blot analysis. Biochemical markers were analyzed in culture media. Apoptosis was studied by immunofluorescence staining and western blots for activated caspase-3. RESULTS: Among the three metabolites tested, 2-MCA consistently produced the most pronounced effects. Exposure to 2-MCA caused morphological changes in neuronal and glial cells already at 0.01 mM. At the biochemical level the most striking result was a significant ammonium increase in culture media with a concomitant glutamine decrease. Dose-response studies showed significant and parallel changes of ammonium and glutamine starting from 0.1 mM 2-MCA. An increased apoptosis rate was observed by activation of caspase-3 after exposure to at least 0.1 mM 2-MCA. CONCLUSION: Surprisingly, 2-MCA, and not MMA, seems to be the most toxic metabolite in our in vitro model leading to delayed axonal growth, apoptosis of glial cells and to unexpected ammonium increase. Morphological changes were already observed at 2-MCA concentrations as low as 0.01 mM. Increased apoptosis and ammonium accumulation started at 0.1 mM thus suggesting that ammonium accumulation is secondary to cell suffering and/or cell death. Local accumulation of ammonium in CNS, that may remain undetected in plasma and urine, may therefore play a key role in the neuropathogenesis of methylmalonic aciduria both during acute decompensations and in chronic phases. If confirmed in vivo, this finding might shift the current paradigm and result in novel therapeutic strategies.

A MODEL OF CREATINE DEFICIENCY SYNDROMES IN 3D BRAIN CELL CULTURES BY KNOCKDOWN OF GAMT AND SLC6A8 GENES

La créatine joue un rôle essentiel dans le métabolisme cellulaire par sa conversion, par la creatine kinase, en phosphocreatine permettant la régénération de l'ATP. La synthèse de créatine, chez les mammifères, s'effectue par une réaction en deux étapes impliquant Γ arginine: glycine amidinotransférase (AGAT) et la guanidinoacétate méthyltransférase (GAMT). L'entrée de créatine dans les cellules s'effectue par son transporteur, SLC6A8. Les déficiences en créatine, dues au déficit en GAMT, AGAT ou SLC6A8, sont fréquentes et caractérisées par une absence ou une forte baisse de créatine dans le système nerveux central. Alors qu'il est connu que AGAT, GAMT et SLC6A8 sont exprimés par le cerveau, les conséquences des déficiences en créatine sur les cellules nerveuses sont peu comprises. Le but de ce travail était de développer de nouveaux modèles expérimentaux des déficiences en Cr dans des cultures 3D de cellules nerveuses de rat en agrégats au moyen de l'interférence à l'ARN appliquée aux gènes GAMT et SLC6A8. Des séquences interférentes (shRNAs) pour les gènes GAMT et SLC6A8 ont été transduites par des vecteurs viraux AAV (virus adéno-associés), dans les cellules nerveuses en agrégats. Nous avons ainsi démontré une baisse de l'expression de GAMT au niveau protéique (mesuré par western blot), et ARN messager (mesuré par qPCR) ainsi qu'une variation caractérisitique de créatine et guanidinoacétate (mesuré par spectrométrie de masse). Après avoir validé nos modèles, nous avons montré que les knockdown de GAMT ou SLC6A8 affectent le développement des astrocytes et des neurones ou des oligodendrocytes et des astrocytes, respectivement, ainsi qu'une augmentation de la mort cellulaire et des modifications dans le pattern d'activation des voies de signalisation impliquant caspase 3 et p38 MAPK, ayant un rôle dans le processus d'apoptose. - Creatine plays essential roles in energy metabolism by the interconversion, by creatine kinase, to its phosphorylated analogue, phosphocreatine, allowing the regeneration of ATP. Creatine is synthesized in mammals by a two step mechanism involving arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT). Creatine is taken up by cells by a specific transporter, SLC6A8. Creatine deficiency syndromes, due to defects in GAMT, AGAT and SLC6A8, are among the most frequent inborn errors of metabolism, and are characterized by an absence or a severe decrease of creatine in central nervous system, which is the main tissue affected. While it is known that AGAT, GAMT and SLC6A8 are expressed in CNS, many questions remain on the specific effects of AGAT, GAMT and SLC6A8 deficiencies on brain cells. Our aim was to develop new experimental models of creatine deficiencies by knockdown of GAMT and SLC6A8 genes by RNAi in 3D organotypic rat brain cell cultures in aggregates. Specific shRNAs for the GAMT and SLC6A8 genes were transduced in brain cell aggregates by adeno-associated viruses (AAV). The AAV-transduced shRNAs were able to efficiently knockdown the expression of our genes of interest, as shown by a strong decrease of protein by western blotting, a decrease of mRNA by qPCR or characteristic variations of creatine and guanidinoacetate by tandem mass spectrometry. After having validated our experimental models, we have also shown that GAMT and SLC6A8 knockdown affected the development of astrocytes and neurons or oligodendrocytes and astrocytes, respectively. We also observed an increase of cell death and variations in activation pattern of caspase 3 and p38 MAPK pathways, involved in apoptosis, in our experimental model.

JNK3 is abundant in insulin-secreting cells and protects against cytokine-induced apoptosis.

AIMS/HYPOTHESIS: In insulin-secreting cells, activation of the c-Jun NH(2)-terminal kinase (JNK) pathway triggers apoptosis. Whereas JNK1 and JNK2 are ubiquitously produced, JNK3 has been described exclusively in neurons. This report aims to characterise the expression and role in apoptosis of the three JNK isoforms in insulin-secreting cells exposed to cytokines. METHODS: Sections of human and mouse pancreases were used for immunohistochemistry studies with isoform-specific anti-JNK antibodies. Human, pig, mouse and rat pancreatic islets were isolated by enzymatic digestion and RNA or protein extracts were prepared. RNA and protein levels were determined by quantitative RT-PCR and western blotting respectively, using JNK-isoform-specific primers and isoform-specific antibodies; activities of the three JNK isoforms were determined by kinase assays following quantitative immunoprecipitation/depletion of JNK3. JNK silencing was performed with small interfering RNAs and apoptotic rates were determined in INS-1E cells by scoring cells displaying pycnotic nuclei. RESULTS: JNK3 and JNK2 mRNAs are the predominant isoforms expressed in human pancreatic islets. JNK3 is nuclear while JNK2 is also cytoplasmic. In INS-1E cells, JNK3 knockdown increases c-Jun levels and caspase-3 cleavage and sensitises cells to cytokine-induced apoptosis; in contrast, JNK1 or JNK2 knockdown is protective. CONCLUSIONS/INTERPRETATION: In insulin-secreting cells, JNK3 plays an active role in preserving pancreatic beta cell mass from cytokine attacks. The specific localisation of JNK3 in the nucleus, its recruitment by cytokines, and its effects on key transcription factors such as c-Jun, indicate that JNK3 is certainly an important player in the transcriptional control of genes expressed in insulin-secreting cells.

Fas ligand-induced proinflammatory transcriptional responses in reconstructed human epidermis. Recruitment of the epidermal growth factor receptor and activation of MAP kinases.

Fas ligand (FasL) exerts potent proapoptotic and proinflammatory actions on epidermal keratinocytes and has been implicated in the pathogenesis of eczema, toxic epidermal necrolysis, and drug-induced skin eruptions. We used reconstructed human epidermis to investigate the mechanisms of FasL-induced inflammatory responses and their relationships with FasL-triggered caspase activity. Caspase activity was a potent antagonist of the pro-inflammatory gene expression triggered by FasL prior to the onset of cell death. Furthermore, we found that FasL-stimulated autocrine production of epidermal growth factor receptor (EGFR) ligands, and the subsequent activation of EGFR and ERK1 and ERK2 mitogen-activated protein kinases, were obligatory extracellular steps for the FasL-induced expression of a subset of inflammatory mediators, including CXCL8/interleukin (IL)-8, ICAM-1, IL-1alpha, IL-1beta, CCL20/MIP-3alpha, and thymic stromal lymphopoietin. These results expand the known physiological role of EGFR and its ligands from promoting keratinocyte mitogenesis and survival to mediating FasL-induced epidermal inflammation.

Zebrafish Based Small Molecule Screening For Retinoblastoma Treatment.

2320 composés chimiques ont été screenés à l'aide d'une lignée transgénique de zébrafish. Cette lignée comportait un gène humain fortement exprimé très tôt dans le développement et la croissance de différentes tumeurs, dont celle du rétinoblastome. L'activation de ce gène induisait la mort des embryons de lâ lignée transgénique. Nous avons donc pu identifier des composés agissant sur l'effet létal de ce gène. Cette étude a permis d'isoler plusieurs composés dont 1 très intéressant, l'Amitriptyline. Ce composé induit une inhibition de la prolifération et une induction d'apoptose dans les cellules humaines de rétinoblastome mais également dans d'autres cellules cancéreuses dont les ostéosarcomes connus. L'ostéosarçome est connu pour faire partie des cancers secondaires dû au rétinoblastome dans la forme héréditaire notamment. Ce composé induit la survie des embryons et réduit également le niveau d'expression de la protéine humaine intégrée dans la lignée de poisson zèbre transgénique de 50%. Le niveau d'expression de ce gène est également réduit de 50 à 60% dans des cultures cellulaire de rétinoblastome humain. L'inhibition de la prolifération a été démontrée par la réduction d'ATP dans plusieurs lignées cellulaires lorsque celles-ci sont traitées avec ce composé. L'induction d'apoptose a été démontrée par induction 10 fois plus élevée des éléments pro-apoptotiques caspase-3 et caspase-7 ainsi que par l'augmentation 10 fois plus élevée d'un élément anti-apoptotique bcl-2. Ces résultats permettent de croire que ce composé pourrait être utilisé pour traiter le rétinoblastome humain. -- Background: Retinoblastoma is a rare malignant tumor. This disease is the most prevalent intraocular cancer in childhood with an incidence of 1 in 15,000 live births. Many therapies are available to treat retinoblastoma, but best treatments are individually selected according to cases. Cryotherapy, thermotherapy, laser therapy including brachytherapy, radiation therapy and chemotherapy are some examples. New drug and new treatments discovery is essential for cancer therapy including retinoblastoma. Purpose: A vertebrate model as zebrafish for retinoblastoma would provide many advantages especially to perform drug screening. The high number of fertilized eggs per mating, the rapidity of extra¬utero development, the available genetic manipulation, the easy manipulations under a microscope, the ability to dispense embryos in 96-well plates and the direct incubation of chemical compounds in fish water are some examples of the advantages. Therefore, we design a transgenic zebrafish carrying a human gene implicated in retinoblastoma development and maintenance. Results: With the small compounds screening, several compounds were isolated. One of these compounds, Amitriptyline demonstrated proliferation inhibition and apoptosis in human retinoblastoma cells, U20S osteosarcoma cells and MBA-231 breast cancer cells. Osteosarcoma is known as secondary cancer due to retinoblastoma. Amitriptyline induced survival in our zebrafish transgenic line and 50% reduction of the integrated gene expression. In retinoblastoma cultured cells, the expression of this gene was also reduced in a range of 50-60 %. Proliferation inhibition was demonstrated by ATP luminescence assay. Apoptosis was demonstrated by a 10-fold induction of caspase-3 and caspase-7, two pro-apoptotic elements and by a 10-fold reduction of bcl-2 anti-apoptotic element. Conclusion: The results suggest that Amitriptyline could be used to treat human retinoblastoma in the near future.

Antigen Receptor Signaling to NF-kappa B via CARMA1, BCL10, and MALT1

The signaling pathway controlling antigen receptor-induced regulation of the transcription factor NF-kappa B plays a key role in lymphocyte activation and development and the generation of lymphomas. Work of the past decade has led to dramatic progress in the identification and characterization of new players in the pathway. Moreover, novel enzymatic activities relevant for this pathway have been discovered, which represent interesting drug targets for immuno-suppression or lymphoma treatment. Here, we summarize these findings and give an outlook on interesting open issues that need to be addressed in the future.

Beclin 1-independent autophagy contributes to apoptosis in cortical neurons.

Neuronal autophagy is enhanced in many neurological conditions, such as cerebral ischemia and traumatic brain injury, but its role in associated neuronal death is controversial, especially under conditions of apoptosis. We therefore investigated the role of autophagy in the apoptosis of primary cortical neurons treated with the widely used and potent pro-apoptotic agent, staurosporine (STS). Even before apoptosis, STS enhanced autophagic flux, as shown by increases in autophagosomal (LC3-II level, LC3 punctate labeling) and lysosomal (cathepsin D, LAMP1, acid phosphatase, β-hexasominidase) markers. Inhibition of autophagy by 3-methyladenine, or by lentivirally-delivered shRNAs against Atg5 and Atg7, strongly reduced the STS-induced activation of caspase-3 and nuclear translocation of AIF, and gave partial protection against neuronal death. Pan-caspase inhibition with Q-VD-OPH likewise protected partially against neuronal death, but failed to affect autophagy. Combined inhibition of both autophagy and caspases gave strong synergistic neuroprotection. The autophagy contributing to apoptosis was Beclin 1-independent, as shown by the fact that Beclin 1 knockdown failed to reduce it but efficiently reduced rapamycin-induced autophagy. Moreover the Beclin 1 knockdown sensitized neurons to STS-induced apoptosis, indicating a cytoprotective role of Beclin 1 in cortical neurons. Caspase-3 activation and pyknosis induced by two other pro-apoptotic stimuli, MK801 and etoposide, were likewise found to be associated with Beclin 1-independent autophagy and reduced by the knockdown of Atg7 but not Beclin 1. In conclusion, Beclin 1-independent autophagy is an important contributor to both the caspase-dependent and -independent components of neuronal apoptosis and may be considered as an important therapeutic target in neural conditions involving apoptosis.

Equine herpesvirus-2 E10 gene product, but not its cellular homologue, activates NF-kappaB transcription factor and c-Jun N-terminal kinase.

We have previously reported on the death effector domain containing E8 gene product from equine herpesvirus-2, designated FLICE inhibitory protein (v-FLIP), and on its cellular homologue, c-FLIP, which inhibit the activation of caspase-8 by death receptors. Here we report on the structure and function of the E10 gene product of equine herpesvirus-2, designated v-CARMEN, and on its cellular homologue, c-CARMEN, which contain a caspase-recruiting domain (CARD) motif. c-CARMEN is highly homologous to the viral protein in its N-terminal CARD motif but differs in its C-terminal extension. v-CARMEN and c-CARMEN interact directly in a CARD-dependent manner yet reveal different binding specificities toward members of the tumor necrosis factor receptor-associated factor (TRAF) family. v-CARMEN binds to TRAF6 and weakly to TRAF3 and, upon overexpression, potently induces the c-Jun N-terminal kinase (JNK), p38, and nuclear factor (NF)-kappaB transcriptional pathways. c-CARMEN or truncated versions thereof do not appear to induce JNK and NF-kappaB activation by themselves, nor do they affect the JNK and NF-kappaB activating potential of v-CARMEN. Thus, in contrast to the cellular homologue, v-CARMEN may have additional properties in its unique C terminus that allow for an autonomous activator effect on NF-kappaB and JNK. Through activation of NF-kappaB, v-CARMEN may regulate the expression of the cellular and viral genes important for viral replication.

The scaffold protein IB1/JIP-1 is a critical mediator of cytokine-induced apoptosis in pancreatic beta cells.

In insulin-secreting cells, cytokines activate the c-Jun N-terminal kinase (JNK), which contributes to a cell signaling towards apoptosis. The JNK activation requires the presence of the murine scaffold protein JNK-interacting protein 1 (JIP-1) or human Islet-brain 1(IB1), which organizes MLK3, MKK7 and JNK for proper signaling specificity. Here, we used adenovirus-mediated gene transfer to modulate IB1/JIP-1 cellular content in order to investigate the contribution of IB1/JIP-1 to beta-cell survival. Exposure of the insulin-producing cell line INS-1 or isolated rat pancreatic islets to cytokines (interferon-gamma, tumor necrosis factor-alpha and interleukin-1beta) induced a marked reduction of IB1/JIP-1 content and a concomitant increase in JNK activity and apoptosis rate. This JNK-induced pro-apoptotic program was prevented in INS-1 cells by overproducing IB1/JIP-1 and this effect was associated with inhibition of caspase-3 cleavage. Conversely, reducing IB1/JIP-1 content in INS-1 cells and isolated pancreatic islets induced a robust increase in basal and cytokine-stimulated apoptosis. In heterozygous mice carrying a selective disruption of the IB1/JIP-1 gene, the reduction in IB1/JIP-1 content in happloinsufficient isolated pancreatic islets was associated with an increased JNK activity and basal apoptosis. These data demonstrate that modulation of the IB1-JIP-1 content in beta cells is a crucial regulator of JNK signaling pathway and of cytokine-induced apoptosis.

Apoptotic markers in protozoan parasites.

ABSTRACT: The execution of the apoptotic death program in metazoans is characterized by a sequence of morphological and biochemical changes that include cell shrinkage, presentation of phosphatidylserine at the cell surface, mitochondrial alterations, chromatin condensation, nuclear fragmentation, membrane blebbing and the formation of apoptotic bodies. Methodologies for measuring apoptosis are based on these markers. Except for membrane blebbing and formation of apoptotic bodies, all other events have been observed in most protozoan parasites undergoing cell death. However, while techniques exist to detect these markers, they are often optimised for metazoan cells and therefore may not pick up subtle differences between the events occurring in unicellular organisms and multi-cellular organisms.In this review we discuss the markers most frequently used to analyze cell death in protozoan parasites, paying special attention to changes in cell morphology, mitochondrial activity, chromatin structure and plasma membrane structure/permeability. Regarding classical regulators/executors of apoptosis, we have reviewed the present knowledge of caspase-like and nuclease activities.

Interaction of Leukocyte Elastase Inhibitor/L-DNase II with BCL-2 and BAX

Leukocyte Elastase Inhibitor (LEI, also called serpin B1) is a protein involved in apoptosis among other physiological processes. We have previously shown that upon cleavage by its cognate protease, LEI is transformed into L-DNase II, a protein with a pro-apoptotic activity. The caspase independent apoptotic pathway, in which L-DNase II is the final effector, interacts with other pro-apoptotic molecules like Poly-ADP-Ribose polymerase (PARP) or Apoptosis Inducing Factor (AIF). The screening of LEI/L-DNase II interactions showed a possible interaction with several members of the BCL-2 family of proteins which are known to have a central role in the regulation of caspase dependent cell death. In this study, we investigated the regulation of LEI/L-DNase II pathway by two members of this family of proteins: BAX and BCL-2, which have opposite effects on cell survival. We show that, in both BHK and HeLa cells, LEI/L-DNase II can interact with BCL-2 and BAX in apoptotic and non-apoptotic conditions. These proteins which are usually thought to be anti-apoptotic and pro-apoptotic respectively, both inhibit the L-DNase II pro-apoptotic activity. These results give further insight in the regulation of caspase-independent pathways and highlight the involvement of the intracellular environment of a given protein in the determinism of its function. They also add a link between caspase-dependent and independent pathways of apoptosis.

Postischemic treatment of neonatal cerebral ischemia should target autophagy.

OBJECTIVE: To evaluate the contributions of autophagic, necrotic, and apoptotic cell death mechanisms after neonatal cerebral ischemia and hence define the most appropriate neuroprotective approach for postischemic therapy. METHODS: Rats were exposed to transient focal cerebral ischemia on postnatal day 12. Some rats were treated by postischemic administration of pan-caspase or autophagy inhibitors. The ischemic brain tissue was studied histologically, biochemically, and ultrastructurally for autophagic, apoptotic, and necrotic markers. RESULTS: Lysosomal and autophagic activities were increased in neurons in the ischemic area from 6 to 24 hours postinjury, as shown by immunohistochemistry against lysosomal-associated membrane protein 1 and cathepsin D, by acid phosphatase histochemistry, by increased expression of autophagosome-specific LC3-II and by punctate LC3 staining. Electron microscopy confirmed the presence of large autolysosomes and putative autophagosomes in neurons. The increases in lysosomal activity and autophagosome formation together demonstrate increased autophagy, which occurred mainly in the border of the lesion, suggesting its involvement in delayed cell death. We also provide evidence for necrosis near the center of the lesion and apoptotic-like cell death in its border, but in nonautophagic cells. Postischemic intracerebroventricular injections of autophagy inhibitor 3-methyladenine strongly reduced the lesion volume (by 46%) even when given >4 hours after the beginning of the ischemia, whereas pan-caspase inhibitors, carbobenzoxy-valyl-alanyl-aspartyl(OMe)-fluoromethylketone and quinoline-val-asp(OMe)-Ch2-O-phenoxy, provided no protection. INTERPRETATION: The prominence of autophagic neuronal death in the ischemic penumbra and the neuroprotective efficacy of postischemic autophagy inhibition indicate that autophagy should be a primary target in the treatment of neonatal cerebral ischemia.

Targeting essential pathways in trypanosomatids gives insights into protozoan mechanisms of cell death.

Apoptosis is a normal component of the development and health of multicellular organisms. However, apoptosis is now considered a prerogative of unicellular organisms, including the trypanosomatids of the genera Trypanosoma spp. and Leishmania spp., causative agents of some of the most important neglected human diseases. Trypanosomatids show typical hallmarks of apoptosis, although they lack some of the key molecules contributing to this process in metazoans, like caspase genes, Bcl-2 family genes and the TNF-related family of receptors. Despite the lack of these molecules, trypanosomatids appear to have the basic machinery to commit suicide. The components of the apoptotic execution machinery of these parasites are slowly coming into light, by targeting essential processes and pathways with different apoptogenic agents and inhibitors. This review will be confined to the events known to drive trypanosomatid parasites to apoptosis.