Synapse formation on adult-born hippocampal neurons.

It is now widely accepted that adult neurogenesis plays a fundamental role in hippocampal function. Neurons born in the adult dentate gyrus of the hippocampus undergo a series of events before they fully integrate in the network and eventually become undistinguishable from neurons born during embryogenesis. Adult hippocampal neurogenesis is strongly regulated by neuronal activity and neurotransmitters, and the synaptic integration of adult-born neurons occurs in discrete steps, some of which are very different from perinatal synaptogenesis. Here, we review the current knowledge on the development of the synaptic input and output of neurons born in the adult hippocampus, from the stem/progenitor cell to the fully mature neuron. We also provide insight on the regulation of adult neurogenesis by some neurotransmitters and discuss some specificities of the integration of new neurons in an adult environment. The understanding of the mechanisms regulating the synaptic integration of adult-born neurons is not only crucial for our understanding of brain plasticity, but also provides a framework for the manipulation and monitoring of endogenous adult neurogenesis as well as grafted cells, for potential therapeutic applications.

VEGF and TNF up-regulate, NSAID down-regulate SOX18 protein level in HUVEC

Angiogenesis, the process of generating new blood vessels, is essential to embryonic development, organ formation, tissue regeneration and remodeling, reproduction and wound healing. Also, it plays an important role in many pathological conditions, including chronic inflammation and cancer. Angiogenesis is regulated by a complex interplay of growth factors, inflammatory mediators, adhesion molecules, morphogens and guidance molecules. Transcription factor SOX18 is transiently expressed in nascent endothelial cells during embryonic development and postnatal angiogenesis, but little is known about signaling pathways controlling its expression. The aim of this study was to investigate whether pro-angiogenic molecules and pharmacological inhibitors of angiogenesis modulate SOX18 expression in endothelial cells. Therefore, we treated human umbilical vein endothelial cells (HUVEC) with angiogenic factors, extracellular matrix proteins, inflammatory cytokines and nonsteroidal anti-inflammatory drugs (NSAID) and monitored SOX18 expression. We have observed that the angiogenic factor VEGF and the inflammatory cytokine TNF increase, while the NSAID ibuprofen and NS398 decrease the SOX18 protein level. These results for the first time demonstrate that SOX18 expression is modulated by factors and drugs known to positively or negatively regulate angiogenesis. This opens the possibility of pharmacological manipulation of SOX18 gene expression in endothelial cells to stimulate or inhibit angiogenesis.

Pseudomonas aeruginosa adhesion to normal and injured respiratory mucosa

Human nasal polyps outgrowth culture were used to study the adhesion of Pseudomonas aeruginosa to respiratory cells. By transmission electron microscopy, bacteria associated with ciliated cells were identified trapped at the extremities of cilia, usually as aggregates of several bacterial cells. They were never seen at the interciliary spaces or attached along cilia. Bacteria were also seen to adhere to migrating cells of the periphery of the outgrowth culture. Using a model of repair of wounded respiratory epithelial cells in culture, we observed that the adhesion of P. aeruginosa to migrating cells of the edges of the repairing wounds was significantly higher than the adhesion to non-migrating cells and that adherent bacteria were surrounded by a fibrocnectin-containing fibrillar material The secretion of extracellular matrix components is involved in the process of epithelium repair following injury. To investigate the molecular basis of P. aeruginosa adhesion to migrating cells, bacteria were treated with a fibronectin solution before their incubation with the respiratory cells. P. aeruginosa treatment by fibronectin significantly increased their adhesion to migrating cells. Accordingly, we hypothesize that during cell migration, fibronectin secreted by epithelial cells may favour P. aeruginosa adhesion by establishing a bridge between the bacteria and the epithelial cell receptors. Such a mechanism may represent a critical step for P. aeruginosa infection of healing injured epithelium.

Low doses of ionizing radiation promote tumor growth and metastasis by enhancing angiogenesis.

Radiotherapy is a widely used treatment option in cancer. However, recent evidence suggests that doses of ionizing radiation (IR) delivered inside the tumor target volume, during fractionated radiotherapy, can promote tumor invasion and metastasis. Furthermore, the tissues that surround the tumor area are also exposed to low doses of IR that are lower than those delivered inside the tumor mass, because external radiotherapy is delivered to the tumor through multiple radiation beams, in order to prevent damage of organs at risk. The biological effects of these low doses of IR on the healthy tissue surrounding the tumor area, and in particular on the vasculature remain largely to be determined. We found that doses of IR lower or equal to 0.8 Gy enhance endothelial cell migration without impinging on cell proliferation or survival. Moreover, we show that low-dose IR induces a rapid phosphorylation of several endothelial cell proteins, including the Vascular Endothelial Growth Factor (VEGF) Receptor-2 and induces VEGF production in hypoxia mimicking conditions. By activating the VEGF Receptor-2, low-dose IR enhances endothelial cell migration and prevents endothelial cell death promoted by an anti-angiogenic drug, bevacizumab. In addition, we observed that low-dose IR accelerates embryonic angiogenic sprouting during zebrafish development and promotes adult angiogenesis during zebrafish fin regeneration and in the murine Matrigel assay. Using murine experimental models of leukemia and orthotopic breast cancer, we show that low-dose IR promotes tumor growth and metastasis and that these effects were prevented by the administration of a VEGF receptor-tyrosine kinase inhibitor immediately before IR exposure. These findings demonstrate a new mechanism to the understanding of the potential pro-metastatic effect of IR and may provide a new rationale basis to the improvement of current radiotherapy protocols.

Cooperative expression of junctional adhesion molecule-C and -B supports growth and invasion of glioma.

Brain invasion is a biological hallmark of glioma that contributes to its aggressiveness and limits the potential of surgery and irradiation. Deregulated expression of adhesion molecules on glioma cells is thought to contribute to this process. Junctional adhesion molecules (JAMs) include several IgSF members involved in leukocyte trafficking, angiogenesis, and cell polarity. They are expressed mainly by endothelial cells, white blood cells, and platelets. Here, we report JAM-C expression by human gliomas, but not by their normal cellular counterpart. This expression correlates with the expression of genes involved in cytoskeleton remodeling and cell migration. These genes, identified by a transcriptomic approach, include poliovirus receptor and cystein-rich 61, both known to promote glioma invasion, as well as actin filament associated protein, a c-Src binding partner. Gliomas also aberrantly express JAM-B, a high affinity JAM-C ligand. Their interaction activates the c-Src proto-oncogene, a central upstream molecule in the pathways regulating cell migration and invasion. In the tumor microenvironment, this co-expression may thus promote glioma invasion through paracrine stimuli from both tumor cells and endothelial cells. Accordingly, JAM-C/B blocking antibodies impair in vivo glioma growth and invasion, highlighting the potential of JAM-C and JAM-B as new targets for the treatment of human gliomas.

Hard-wired heterogeneity in blood stem cells revealed using a dynamic regulatory network model.

MOTIVATION: Combinatorial interactions of transcription factors with cis-regulatory elements control the dynamic progression through successive cellular states and thus underpin all metazoan development. The construction of network models of cis-regulatory elements, therefore, has the potential to generate fundamental insights into cellular fate and differentiation. Haematopoiesis has long served as a model system to study mammalian differentiation, yet modelling based on experimentally informed cis-regulatory interactions has so far been restricted to pairs of interacting factors. Here, we have generated a Boolean network model based on detailed cis-regulatory functional data connecting 11 haematopoietic stem/progenitor cell (HSPC) regulator genes. RESULTS: Despite its apparent simplicity, the model exhibits surprisingly complex behaviour that we charted using strongly connected components and shortest-path analysis in its Boolean state space. This analysis of our model predicts that HSPCs display heterogeneous expression patterns and possess many intermediate states that can act as 'stepping stones' for the HSPC to achieve a final differentiated state. Importantly, an external perturbation or 'trigger' is required to exit the stem cell state, with distinct triggers characterizing maturation into the various different lineages. By focusing on intermediate states occurring during erythrocyte differentiation, from our model we predicted a novel negative regulation of Fli1 by Gata1, which we confirmed experimentally thus validating our model. In conclusion, we demonstrate that an advanced mammalian regulatory network model based on experimentally validated cis-regulatory interactions has allowed us to make novel, experimentally testable hypotheses about transcriptional mechanisms that control differentiation of mammalian stem cells. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Visualització de la interaccio entre la proteïnaG12 i la catenina p120

Els processos de senyalització a través de receptors acoplats a proteïnes G (GPCRs) estan implicats en una gran varietat de processos fisiològics i patològics. Els objectius de la meva recerca es centren en l’estudi de la funció de les proteïnes heterotrimèriques de la família G12, en particular, en el paper que aquestes proteïnes poden tenir en la inducció de la migració cel•lular. Des del seu descobriment, s’han descrit diversos efectors que s’uneixen i es regulen per aquestes proteïnes. Les proteïnes de la família de RhoGEF semblen ser els efectors més directes i que juguen un paper més important en els processos de senyalització de les proteïnes G12. Tanmateix, resultats recents semblen indicar que altres vies independent de l’activació de Rho són també necessàries perquè els efectes fisiològics de les vies de les proteïnes G12 tinguin lloc. En aquest camp, els resultats que he obtingut, juntament amb resultats previs del grup, han descrit una nova via d’activació independent de Rho. Hem trobat que la proteïna G12 s’uneix a una catenina: la catenina p120. La seva unió sembla tenir lloc a través l’extrem N-terminal de la catenina i condueix a la reducció de la fosforilació en algun dels seus residus de tirosina, ja sigui per la quinasa Src o per l’activació a través d’EGF. Per tant, aquests resultats suggereixen que una altra via d’acció de la proteïna G12 seria mitjançant la regulació de la catenina p120 i, com a conseqüència, alguns processos d’adhesió cel•lular es podrien veure afectats. A fi d’entendre millor la regulació i la interacció de la catenina p120 hem iniciat l’estudi de la seva distribució cel•lular en l’espai i temps mitjançant tècniques de microscopia. Així, vam intentar construir una sonda de G12 fluorescent amb GFP. Després de diversos intents i experiments amb proteïnes no funcionals hem aconseguit, amb la col•laboració de T. Meigs, una construcció funcional que activa Rho. Això ens permetrà acabar els experiments de seguiment in vivo.

Cathepsin X cleavage of the beta2 integrin regulates talin-binding and LFA-1 affinity in T cells.

T cell migration, essential for immune surveillance and response, is mediated by the integrin LFA-1. CatX, a cysteine carboxypeptidase, is involved in the regulation of T cell migration by interaction with LFA-1. We show that sequential cleavage of C-terminal amino acids from the β(2) cytoplasmic tail of LFA-1, by CatX, enhances binding of the adaptor protein talin to LFA-1 and triggers formation of the latter's high-affinity form. As shown by SPR analysis of peptides constituting the truncated β(2) tail, the cleavage of three C-terminal amino acids by CatX resulted in a 1.6-fold increase of talin binding. Removal of one more amino acid resulted in a 2.5-fold increase over the intact tail. CatX cleavage increased talin-binding affinity to the MD but not the MP talin-binding site on the β(2) tail. This was shown by molecular modeling of the β(2) tail/talin F3 complex to be a result of conformational changes affecting primarily the distal-binding site. Analysis of LFA-1 by conformation-specific mAb showed that CatX modulates LFA-1 affinity, promoting formation of high-affinity from intermediate-affinity LFA-1 but not the initial activation of LFA-1 from a bent to extended form. CatX post-translational modifications may thus represent a mechanism of LFA-1 fine-tuning that enables the trafficking of T cells.

Location of corneal epithelial stem cells Reply

The longstanding concept that corneal epithelial stem cells reside mainly in the limbus is supported by the absence of major corneal epithelial differentiation markers, that is, K3 and K12 keratins, in limbal basal cells (these markers are expressed, however, in corneal basal cells, thus distinguishing the mode of keratin expression in corneal epithelium from that of all other stratified epithelia), the centripetal migration of corneal epithelial cells, the exclusive location of slow-cycling cells in the limbal basal layer, the superior in vitro proliferative potential of limbal epithelial cells, and the transplanted limbal cells' ability to reconstitute corneal epithelium in vivo (reviewed in refs 1-4). Moreover, previous data indicate that corneal and conjunctival epithelia represent two separate cell lineages (reviewed in refs 1-4). Majo et al. suggested, however, that corneal and conjunctival epithelia are equipotent, and that identical oligopotent stem cells are present throughout the corneal, limbal and conjunctival epithelia. We point out here that these suggestions are inconsistent with many known growth, differentiation and cell migration properties of the anterior ocular epithelia.

Influence of epidermal growth factor on the maturation of fetal rat brain cells in aggregate culture. An immunocytochemical study.

Maturation of astrocytes, neurons, and oligodendrocytes was studied in serum-free aggregating cell cultures of fetal rat telencephalon by an immunocytochemical approach. Cell type-specific immunofluorescence staining was examined by using antibodies directed against glial fibrillary acidic protein (GFAP) and vimentin, two astroglial markers; neuron-specific enolase (NSE) and neurofilament (NF), two neuronal markers, and galactocerebroside (GC), an oligodendroglial marker. It was found that the cellular maturation in aggregates is characterized by distinct developmental increases in immunoreactivity for GFAP, vimentin, NSE, NF, and GC, and by a subsequent decrease of vimentin-positive structures in more differentiated cultures. These findings are in agreement with observations in vivo, and they corroborate previous biochemical studies of this histotypic culture system. Treatment of very immature cultures with a low dose of epidermal growth factor (EGF, 5 ng/ml) enhanced the developmental increase in GFAP, NSE, NF and GC immunoreactivity, suggesting an acceleration of neuronal and glial maturation. In addition, EGF was found to alter the cellular organization within the aggregates, presumably by influencing cell migration.

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.

TNF/TNFR1 signaling up-regulates CCR5 expression by CD8+ T lymphocytes and promotes heart tissue damage during Trypanosoma cruzi infection: beneficial effects of TNF-α blockade

In Chagas disease, understanding how the immune response controls parasite growth but also leads to heart damage may provide insight into the design of new therapeutic strategies. Tumor necrosis factor-alpha (TNF-α) is important for resistance to acute Trypanosoma cruzi infection; however, in patients suffering from chronic T. cruzi infection, plasma TNF-α levels correlate with cardiomyopathy. Recent data suggest that CD8-enriched chagasic myocarditis formation involves CCR1/CCR5-mediated cell migration. Herein, the contribution of TNF-α, especially signaling through the receptor TNFR1/p55, to the pathophysiology of T. cruzi infection was evaluated with a focus on the development of myocarditis and heart dysfunction. Colombian strain-infected C57BL/6 mice had increased frequencies of TNFR1/p55+ and TNF-α+ splenocytes. Although TNFR1-/- mice exhibited reduced myocarditis in the absence of parasite burden, they succumbed to acute infection. Similar to C57BL/6 mice, Benznidazole-treated TNFR1-/- mice survived acute infection. In TNFR1-/- mice, reduced CD8-enriched myocarditis was associated with defective activation of CD44+CD62Llow/- and CCR5+ CD8+ lymphocytes. Also, anti-TNF-α treatment reduced the frequency of CD8+CCR5+ circulating cells and myocarditis, though parasite load was unaltered in infected C3H/HeJ mice. TNFR1-/- and anti-TNF-α-treated infected mice showed regular expression of connexin-43 and reduced fibronectin deposition, respectively. Furthermore, anti-TNF-α treatment resulted in lower levels of CK-MB, a cardiomyocyte lesion marker. Our results suggest that TNF/TNFR1 signaling promotes CD8-enriched myocarditis formation and heart tissue damage, implicating the TNF/TNFR1 signaling pathway as a potential therapeutic target for control of T. cruzi-elicited cardiomyopathy.

Breast cancer suppressor candidate-1 (BCSC-1) is a melanoma tumor suppressor that down regulates MITF.

Understanding the molecular aberrations involved in the development and progression of metastatic melanoma (MM) is essential for a better diagnosis and targeted therapy. We identified breast cancer suppressor candidate-1 (BCSC-1) as a novel tumor suppressor in melanoma. BCSC-1 expression is decreased in human MM, and its ectopic expression in MM-derived cell lines blocks tumor formation in vivo and melanoma cell proliferation in vitro while increasing cell migration. We demonstrate that BCSC-1 binds to Sox10, which down regulates MITF, and results in a switch of melanoma cells from a proliferative to a migratory phenotype. In conclusion, we have identified BCSC-1 as a tumor suppressor in melanoma and as a novel regulator of the MITF pathway.

Genetic investigation of the functions of c-Myc and N-Myc in Hematopoietic stem cells

Résumé : c-Myc, le premier facteur de transcription de la famille Myc a été découvert il y a maintenant trente ans. Il reste à l'heure actuelle parmi les plus puissants proto-oncogènes connus. c-Myc est dérégulé dans plus de 50% des cancers, où il promeut la prolifération, la croissance cellulaire, et la néoangiogenèse. Myc peut aussi influencer de nombreuses autres fonctions de par sa capacité à activer ou à réprimer la transcription de nombreux gènes, et à agir globalement sur le génome à travers des modifications épigénétiques de la chromatine. La famille d'oncogènes Myc comprend, chez les mammifères, trois protéines structurellement proches: c-Myc, N-Myc et L-Myc. Ces protéines ont les mêmes proprietés biochimiques, exercent les mêmes fonctions mais sont le plus souvent exprimées de façon mutuellement exclusive. Myc a été récemment identifié comme un facteur clef dans la maintenance des cellules souches embryonnaires et adultes ainsi que dans la réacquisition des proprietés des cellules souches. Nous avons précédemment démontré que l'élimination de c-Myc provoque une accumulation de cellules souches hématopoïétiques (CSH) suite à un défaut de différenciation lié à la niche. Les CSH sont responsables de la production de tous les éléments cellulaires du sang pour toute la vie de l'individu et sont définies par leur capacité à s'auto-renouveler tout en produisant des précurseurs hématopoïétiques. Afin de mieux comprendre la fonction de Myc dans les CSH, nous avons choisi de combiner l'utilisation de modèles de souris génétiquement modifiées à une caractérisation systématique des schémas d'expression de c-Myc, N-Myc et L-Myc dans tout le système hématopoïétique. Nous avons ainsi découvert que les CSH les plus immatures expriment des quantités équivalentes de transcrits de c-myc et N-myc. Si les CSH déficientes en N-myc seulement ont une capacité d'auto-renouvellement à long-terme réduite, l'invalidation combinée des gènes c-myc et N-myc conduit à une pan-cytopénie suivie d'une mort rapide de l'animal, pour cause d'apoptose de tous les types cellulaires hématopoïétiques. En particulier, les CSH en cours d'auto-renouvelemment, mais pas les CSH quiescentes, accumulent du Granzyme B (GrB), une molécule fortement cytotoxique qui provoque une mort cellulaire rapide. Ces données ont ainsi mis au jour un nouveau mécanisme dont dépend la survie des CSH, à savoir la répression du GrB, une enzyme typiquement utilisée par le système immunitaire inné pour éliminer les tumeurs et les cellules infectées par des virus. Dans le but d'évaluer l'étendue de la redondance entre c-Myc et N-Myc dans les CSH, nous avons d'une part examiné des souris dans lesquelles les séquences codantes de c-myc sont remplacées par celles de N-myc (NCR) et d'autre part nous avons géneré une série allèlique de myc en éliminant de façon combinatoire un ou plusieurs allèles de c-myc et/ou de N-myc. Alors que l'analyse des souris NCR suggère que c-Myc et N-Myc sont qualitativement redondants, la série allélique indique que les efficiences avec lesquelles ces deux protéines influencent des procédés essentiels à la maintenance des CSH sont différentes. En conclusion, nos données génétiques montrent que l'activité générale de MYC, fournie par c-Myc et N-Myc, contrôle plusieurs aspects cruciaux de la fonction des CSH, notamment l'auto-renouvellement, la survie et la différenciation. Abstract : c-Myc, the first Myc transcription factor was discovered 30 years ago and is to date one of the most potent proto-oncogenes described. It is found to be misregulated in over 50% of all cancers, where it drives proliferation, cell growth and neo-angiogenesis. Myc can also influence a variety of other functions, owing to its ability to activate and repress transcription of many target genes and to globally regulate the genome via epigenetic modifications of the chromatin. The Myc family of oncogenes consists of three closely related proteins in mammals: c-Myc, N-Myc and L-Myc. These proteins share the same biochemical properties, exert mostly the same functions, but are most often expressed in mutually exclusive patterns. Myc is now emerging as a key factor in maintenance of embryonic and adult stem cells as well as in reacquisition of stem cell properties, including induced reprogramming. We previously showed that c-Myc deficiency can cause the accumulation of hematopoietic stem cells (HSCs) due to a niche dependent differentiation defect. HSCs are responsible for life-long replenishment of all blood cell types, and are defined by their ability to self-renew while concomitantly giving rise to more commited progenitors. To gain further insight into the function of Myc in HSCs, in this study we combine the use of genetically-modified mouse models with the systematic characterization of c-myc, N-myc and L-myc transcription patterns throughout the hematopoietic system. Interestingly, the most immature HSCs express not only c-myc, but also about equal amounts of N-myc transcripts. Although conditional deletion of N-myc alone in the bone marrow does not affect steady-state hematopoiesis, N-myc null HSCs show impaired long-term self-renewal capacity. Strikingly, combined deficiency of c-Myc and N-Myc results in pan-cytopenia and rapid lethality, due to the apoptosis of most hematopoietic cell types. In particular, self-renewing HSCs, but not quiescent HSCs or progenitor cell types rapidly up-regulate and accumulate the potent cytotoxic molecule GranzymeB (GrB), causing their rapid cell death. These data uncover a novel pathway on which HSC survival depends on, namely repression of GrB, a molecule typically used by the innate immune system to eliminate tumor and virus infected cells. To evaluate the extent of redundancy between c-Myc and N-Myc in HSCs, we examined mice in which c-myc coding sequences are replaced by that of N-myc (NCR) and also generated an allelic series of myc, by combinatorially deleting one or several c-myc and/or N-myc alleles. While the analysis of NCR mice suggests that c-Myc and N-Myc are qualitatively functionally redundant, our allelic series indicates that the efficiencies with which these two proteins affect crucial HSC maintenance processes are likely to be distinct. Collectively, our genetic data show that general "MYC" activity delivered by c-Myc and N-Myc controls crucial aspects of HSC function, including self-renewal, survival and niche dependent differentiation.

A novel study of copy number variations in Hirschsprung disease using the multiple ligation-dependent probe amplification (MLPA) technique.

BACKGROUND Hirschsprung disease (HSCR) is a congenital malformation of the hindgut produced by a disruption in neural crest cell migration during embryonic development. HSCR has a complex genetic etiology and mutations in several genes, mainly the RET proto-oncogene, have been related to the disease. There is a clear predominance of missense/nonsense mutations in these genes whereas copy number variations (CNVs) have been seldom described, probably due to the limitations of conventional techniques usually employed for mutational analysis. METHODS In this study we have aimed to analyze the presence of CNVs in some HSCR genes (RET, EDN3, GDNF and ZFHX1B) using the Multiple Ligation-dependent Probe Amplification (MLPA) approach. RESULTS Two alterations in the MLPA profiles of RET and EDN3 were detected, but a detailed inspection showed that the decrease in the corresponding dosages were due to point mutations affecting the hybridization probes regions. CONCLUSION Our results indicate that CNVs of the gene coding regions analyzed here are not a common molecular cause of Hirschsprung disease. However, further studies are required to determine the presence of CNVs affecting non-coding regulatory regions, as well as other candidate genes.