Epigenetic regulation of human cancer/testis antigen gene, HAGE, in chronic myeloid leukemia.

BACKGROUND AND OBJECTIVES Cancer testis antigens (CTA) provide attractive targets for cancer-specific immunotherapy. Although CTA genes are expressed in some normal tissues, such as the testis, this immunologically protected site lacks MHC I expression and as such, does not present self antigens to T cells. To date, CTA genes have been shown to be expressed in a range of solid tumors via demethylation of their promoter CpG islands, but rarely in chronic myeloid leukemia (CML) or other hematologic malignancies. DESIGN AND METHODS In this study, the methylation status of the HAGE CTA gene promoter was analyzed by quantitative methylation-specific polymerase chain reaction (MSP) and sequencing in four Philadelphia-positive cell lines (TCC-S, K562, KU812 and KYO-1) and in CML samples taken from patients in chronic phase (CP n=215) or blast crisis (BC n=47). HAGE expression was assessed by quantitative reverse transcriptase-polymerase chain reaction. RESULTS The TCC-S cell line showed demethylation of HAGE that was associated with over-expression of this gene. HAGE hypomethylation was significantly more frequent in BC (46%) than in CP (22%) (p=0.01) and was correlated with high expression levels of HAGE transcripts (p<0.0001). Of note, in CP-CML, extensive HAGE hypomethylation was associated with poorer prognosis in terms of cytogenetic response to interferon (p=0.01) or imatinib (p=0.01), molecular response to imatinib (p=0.003) and progression-free survival (p=0.05). INTERPRETATIONS AND CONCLUSION: The methylation status of the HAGE promoter directly correlates with its expression in both CML cell lines and patients and is associated with advanced disease and poor outcome.

Genetic dissection of c-Myc function during mouse organogenesis

Résumé Le gène c-myc est un des oncogènes les plus fréquemment mutés dans les tumeurs humaines. Même si plus de 70 % des cancers humains montrent une dérégulation de c-Myc, les connaissances sur son rôle physiologique pendant le développement, et dans la souris adulte restent très peu connus. Récemment, notre laboratoire a pu montrer que c-Myc contrôle l'équilibre entre le renouvellement et la différenciation des cellules souches hématopoïetiques (CSH) dans la souris adulte. Ceci est probablement dû à lacapacité de c-Myc de contrôler l'entrée et la sortie des CSH de leur niche de la moelle osseuse, en régulant plusieurs molécules d'adhésion, parmi lesquelles la cadhérine-N (Wilson et al., 2004; Wilson and Trumpp, 2006). Des études utilisant un mutant d'inactivation ont demontré que la protéine c-Myc est essentielle pour le développement au delà du jour embryonnaire E9.5. Les embryons c-Myc déficients sont plus petits que la normale et possèdent de nombreux défauts; en particulier ils ne peuvent établir un système hématopoietique embryonnaire primitif (Trumpp et al., 2001). Nous avons récemment découvert que le développement du placenta dépend de la présence de cMyc. Ceci permet de proposer que certains, sinon tous, les défauts embryonnaires puorraient dériver indirectement d'un défaut nutritionnel causé par la défaillance du placenta. Afin de répondre à cette question de manière génétique, nous avons utilisé l'allele conditionel c-mycflox (Trumpp et al., 2001) en combinaison avec l'allele Sox2-Cre (Hayashi et al., 2002). Celui-ci détermine l'expression de la récombinase Cre spécifiquement dans les cellules de l'épiblaste à partir de E6.5, tandis qu'il n'y a pas, ou seulement très peu, d'activité de la récombinase Cre dans les tissus extraembryonnaires.Alnsi, cette stratégie nous permet de générer des embryons sans c-Myc qui se développent en présence d'un compartment extraembryonnaire ou c-Myc est exprimé normalement (Sox2Cre;c-mycflox2) Ces embryons, Sox2Cre;c-mycflox2 se développent et grandissent normalement tout en formant un système vasculaire normal, mais meurent à E11.5 à cause d'un sévère manque de cellules hématopoïetiques. De façon très intéressante, la seule population qui semble être présente en nombre à peu près normal dans ces embryons est celle des précurseurs et des cellules souches. Les cellules qui forment cette population prolifèrent normalement mais ne peuvent pas former des colonies in vitro, ce qui montre que ces cellules ont perdu leur activité de cellules souches. Cependant, lorsque nous avons analysé ces cellules plus en détail en éxaminant l'expression des molécules d'intégrine nous avons découvert que l'integrine ß est sur-éxprimée à la surface des cellules c-Myc déficientes. Ceci pourrait indiquer un mécanisme par lequel c-Myc régule des molécules d'adhésion sur les cellules du sang. En conséquence, en absence de c-Myc, l'adhésion et la migration des cellules du sang de l'AGM (Aorte-Gonade-Mésonéphros) vers le foie de l'embryon, à travers le système vasculaire, est compromise. En outre, nous avons pu montrer que les hépatocytes du foie, qui constitue le site principal de formation des cellules hématopoïetiques pendant le développement, est sévèrement atteint dans des Sox2Cre;c-mycflox2 embryons. Ceci n'est pas du à un défaut propre aux cellules hépatiques qui ont perdu c-Myc, mais résulte plutôt de l'absence de cellules hématopoietïques qui normalement colonisent le foie à ce stade du développement. Ces résultats représentent la première preuve directe que le développement des hépatoblastes est dépendant de signaux provenant des cellules du sang. Summary The myc gene is one of the most frequently mutated oncogenes in human tumors. It is found to be mis-regulated in over 70% of all human cancers. However, our knowledge about its physiological role in mammalian development and adulthood remains limited. Recent work in our laboratory showed that c-Myc controls the balance between hematopoietic stem cell (HSC) self-renewal and differentiation in the adult mouse. This is likely due to the capacity of c-Myc to control entry and exit of HSCs from the bone marrow niche by regulating a number of cell adhesion molecules including N-cadherin (Wilson et al., 2004; Wilson and Trumpp 2006). During development knockout studies showed that c-Myc is required for embryonic development beyond embryonic day (E) 9.5. c-Myc deficient embryos are severely reduced in size and show multiple defects including the failure to establish a primitive hematopoietic system (Trumpp et al., 2001). Importantly, we recentry uncovered that placental development also seems to depend on normal c-Myc function, raising the possibility that some if not all of the embryonic defects observed could be mediated indirectly by a nutrition defect caused by placental failure. To address this possibility genetically, we took advantage of the conditional c-mycflox allele (Trumpp et al., 2001) in combination with the Sox2-Cre allele (Hayashi et al., 2002), in which Cre expression is specifically targeted to all epiblast cells by E6.5, while there is little or no Cre activity inextra-embryonic lineages. Thus, this strategy allows the generation of c-Myc deficient embryos, which develop within a normal c-Myc expressing extra-embryonic compartment (Sox2Cre;c-mycflox2) Such Sox2Cre;c-mycflox2 embryos develop and grow appropriately and form a normal vascular system but die at E11.5 due to a severe lack of blood cells. Interestingly, the only hematopoietic population that seems to be present in almost normal numbers in the embryo is the stem/progenitor cell population. Cells within this populatíon proliferate normal but can not give rise to hematopoietic colonies in vitro showing that functional hematopoietic stem cell (HSC) activity is lost. However, when we analyzed these phenotypic HSCs in more detail and examined integrin expression in mutant stem/progenitor cells, we observed that ß1-integrin is upregulated. This may point to a potential mechanism whereby c-Myc regulates adhesíon molecules on hematopoietic cells and thereby disturbs adhesion and migration from the AGM (aorta-gonads-mesonephros) through the vascular system to the liver. Furthermore, we uncovered that the fetal liver, the main site of hematopoietic expansion at that stage, is severely affected in Sox2Cre;c-mycflox2 embryos and that this is not due to a cell intrinsic defect of c-Myc deficient hepatocytes but rather due to the lack of hematopoietic cells that normally colonize the fetal liver at that stage of development. This provides first direct evidence that hepatoblast development depends on signals derived from blood cells.

Increased condom use without other major changes in sexual behavior among the general population in Switzerland.

The expression of DNA topoisomerase II alpha and beta genes was studied in murine normal tissues. Northern blot analysis using probes specific for the two genes showed that the patterns of expression were different among 22 tissues of adult mice. Expression levels of topoisomerase II alpha gene were high in proliferating tissues, such as bone marrow and spleen, and undetectable or low in 17 other tissues. In contrast, high or intermediate expression of topoisomerase II beta gene was found in a variety of tissues (15) of adult mice, including those with no proliferating cells. Topoisomerase II gene expression was also studied during murine development. In whole embryos both genes were expressed at higher levels in early than late stages of embryogenesis. Heart, brain and liver of embryos two days before delivery, and these same tissues plus lung and thymus of newborn (1-day-old) mice expressed appreciable levels of the two genes. Interestingly, a post-natal induction of the beta gene expression was observed in the brain but not in the liver; conversely, the expression of the alpha gene was increased 1 day after birth in the liver but not in the brain. However, gene expression of a proliferation-associated enzyme, thymidylate synthase, was similar in these tissues between embryos and newborns. Thus, the two genes were differentially regulated in the post-natal period, and a tissue-specific role may be suggested for the two isoenzymes in the development of differentiated tissues such as the brain and liver. Based on the differential patterns of expression of the two isoforms, this analysis indicates that topoisomerase II alpha may be a specific marker of cell proliferation, whereas topoisomerase II beta may be implicated in functions of DNA metabolism other than replication.

Proangiogenic Factor PlGF Programs CD11b+ Myelomonocytes in Breast Cancer during Differentiation of Their Hematopoietic Progenitors.

Tumor-mobilized bone marrow-derived CD11b(+) myeloid cells promote tumor angiogenesis, but how and when these cells acquire proangiogenic properties is not fully elucidated. Here, we show that CD11b(+) myelomonocytic cells develop proangiogenic properties during their differentiation from CD34(+) hematopoietic progenitors and that placenta growth factor (PlGF) is critical in promoting this education. Cultures of human CD34(+) progenitors supplemented with conditioned medium from breast cancer cell lines or PlGF, but not from nontumorigenic breast epithelial lines, generate CD11b(+) cells capable of inducing endothelial cell sprouting in vitro and angiogenesis in vivo. An anti-Flt-1 mAb or soluble Flt-1 abolished the generation of proangiogenic activity during differentiation from progenitor cells. Moreover, inhibition of metalloproteinase activity, but not VEGF, during the endothelial sprouting assay blocked sprouting induced by these proangiogenic CD11b(+) myelomonocytes. In a mouse model of breast cancer, circulating CD11b(+) cells were proangiogenic in the sprouting assays. Silencing of PlGF in tumor cells prevented the generation of proangiogenic activity in circulating CD11b(+) cells, inhibited tumor blood flow, and slowed tumor growth. Peripheral blood of breast cancer patients at diagnosis, but not of healthy individuals, contained elevated levels of PlGF and circulating proangiogenic CD11b(+) myelomonocytes. Taken together, our results show that cancer cells can program proangiogenic activity in CD11b(+) myelomonocytes during differentiation of their progenitor cells in a PlGF-dependent manner. These findings impact breast cancer biology, detection, and treatment. Cancer Res; 71(11); 3781-91. ©2011 AACR.

The Distinct Molecular Signature Of Hepatosplenic T-Cell Lymphoma (Hstl) Identifies Oncogenic Pathways With Potential Therapeutic Relevance

Background: HSTL is a rare entity characterized by an infiltration of bone marrow, spleen and liver tissues by neoplastic gammadelta (gd) -more rarely alphabeta (ab)- T cells. Its pathogenesis is poorly understood. Our purpose was to identify the molecular signature of HSTL and explore molecular pathways implicated in its pathogenesis.Methods: Gene expression profiling and array CGH analysis of 10 HSTL samples (7gd, 3ab), 1 HSTL cell line (DERL2), 2 normal gd samples together with 16 peripheral T-cell lymphoma not otherwise specified (PTCL,NOS) and 7 nasal NK/T cell lymphomas were performed.Results: By unsupervised analysis, ab and gdHSTL clustered together remarkably separated from other lymphoma entities. Compared to PTCL, NOS, HSTL overexpresed genes encoding NK-associated molecules, oncogenes (VAV3) and the Sphingosine-1-phosphatase receptor 5 involved in cell trafficking. Compared to normal gd cells, HSTL overexpressed genes encoding NK-cell and multi drug resistance-associated molecules, transcription factors (RHOB), oncogenes (MAFB, FOS, JUN, VAV3) and the tyrosine kinase SYK whereas genes encoding cytotoxic molecules and the tumor suppressor gene AIM1 were among the most downregulated. By immunohistochemistry, SYK was demonstrated on HSTL cells with expression of its phosphorylated form in DERL2 cells by Western blot. Functional studies using a SYK inhibitor revealed a dose dependent increase of apoptotic DERL2 cells suggesting that SYK could be a candidate target for pharmacologic inhibition. Downexpression of AIM1 was validated by qRT-PCR. Methylation analysis of DERL2 genomic DNA treated by bisulfite demonstrated highly methylated CpG islands of AIM1. Genomic profiles confirmed recurrent isochromosome 7q (n=6/9) without alterations at 9q22 and 6q21 containing SYK and AIM1 genes, respectively.Conclusion: The current study identifies a distinct molecular signature for HSTL and highlights oncogenic pathways which offer rationale for exploring new therapeutic options such as SYK inhibitors. It supports the view of gd and ab HSTL as a single entity.

T cell fate specification and alphabeta/gammadelta lineage commitment.

The development of T cells from pluripotent stem cells involves a coordinated series of lineage-commitment steps. Common lymphoid precursors in the fetal liver or adult bone marrow must first choose between a T, B or NK cell fate. Committed T cell precursors in the thymus then differentiate into cells committed to the alphabeta or gammadelta lineages. Recent advances have been made in our understanding of the mechanisms underlying T cell fate specification and alphabeta/gammadelta lineage divergence.

Modalities and future prospects of gene therapy in heart transplantation.

Heart transplantation is the treatment of choice for many patients with end-stage heart failure. Its success, however, is limited by organ shortage, side effects of immunosuppressive drugs, and chronic rejection. Gene therapy is conceptually appealing for applications in transplantation, as the donor organ is genetically manipulated ex vivo before transplantation. Localised expression of immunomodulatory genes aims to create a state of immune privilege within the graft, which could eliminate the need for systemic immunosuppression. In this review, recent advances in the development of gene therapy in heart transplantation are discussed. Studies in animal models have demonstrated that genetic modification of the donor heart with immunomodulatory genes attenuates ischaemia-reperfusion injury and rejection. Alternatively, bone marrow-derived cells genetically engineered with donor-type major histocompatibility complex (MHC) class I or II promote donor-specific hyporesponsiveness. Genetic engineering of naïve T cells or dendritic cells may induce regulatory T cells and regulatory dendritic cells. Despite encouraging results in animal models, however, clinical gene therapy trials in heart transplantation have not yet been started. The best vector and gene to be delivered remain to be identified. Pre-clinical studies in non-human primates are needed. Nonetheless, the potential of gene therapy as an adjunct therapy in transplantation is essentially intact.

Living-engineered valves for transcatheter venous valve repair.

Background: Chronic venous insufficiency (CVI) represents a major global health problem with increasing prevalence and morbidity. CVI is due to an incompetence of the venous valves, which causes venous reflux and distal venous hypertension. Several studies have focused on the replacement of diseased venous valves using xeno- and allogenic transplants, so far with moderate success due to immunologic and thromboembolic complications. Autologous cell-derived tissue-engineered venous valves (TEVVs) based on fully biodegradable scaffolds could overcome these limitations by providing non-immunogenic, non-thrombogenic constructs with remodeling and growth potential. Methods: Tri- and bicuspid venous valves (n=27) based on polyglycolic acid-poly-4-hydroxybutyrate composite scaffolds, integrated into self-expandable nitinol stents, were engineered from autologous ovine bone-marrow-derived mesenchymal stem cells (BM-MSCs) and endothelialized. After in vitro conditioning in a (flow) pulse duplicator system, the TEVVs were crimped (n=18) and experimentally delivered (n=7). The effects of crimping on the tissue-engineered constructs were investigated using histology, immunohistochemistry, scanning electron microscopy, grating interferometry (GI), and planar fluorescence reflectance imaging. Results: The generated TEVVs showed layered tissue formation with increasing collagen and glycosaminoglycan levels dependent on the duration of in vitro conditioning. After crimping no effects were found on the MSC level in scanning electron microscopy analysis, GI, histology, and extracellular matrix analysis. However, substantial endothelial cell loss was detected after the crimping procedure, which could be reduced by increasing the static conditioning phase. Conclusions: Autologous living small-diameter TEVVs can be successfully fabricated from ovine BM-MSCs using a (flow) pulse duplicator conditioning approach. These constructs hold the potential to overcome the limitations of currently used non-autologous replacement materials and may open new therapeutic concepts for the treatment of CVI in the future.

Protein energy malnutrition increases arginase activity in monocytes and macrophages.

BACKGROUND: Protein energy malnutrition is commonly associated with immune dysfunctions and is a major factor in susceptibility to infectious diseases. METHODS: In this study, we evaluated the impact of protein energy malnutrition on the capacity of monocytes and macrophages to upregulate arginase, an enzyme associated with immunosuppression and increased pathogen replication. RESULTS: Our results show that monocytes and macrophages are significantly increased in the bone marrow and blood of mice fed on a protein low diet. No alteration in the capacity of bone marrow derived macrophages isolated from malnourished mice to phagocytose particles, to produce the microbicidal molecule nitric oxide and to kill intracellular Leishmania parasites was detected. However, macrophages and monocytes from malnourished mice express significantly more arginase both in vitro and in vivo. Using an experimental model of visceral leishmaniasis, we show that following protein energy malnutrition, the increased parasite burden measured in the spleen of these mice coincided with increased arginase activity and that macrophages provide a more permissive environment for parasite growth. CONCLUSIONS: Taken together, these results identify a novel mechanism in protein energy malnutrition that might contributes to increased susceptibility to infectious diseases by upregulating arginase activity in myeloid cells.

Hémato-oncologie pédiatrique: de la biologie cellulaire aux traitements ciblés et individualisés [Pediatric hemato-oncology: from cellular biology to individualized targeted therapies].

Progresses in pediatric oncology over the last decades have been dramatic and allow current cure rates above 80%. There are mainly due to multicentre clinical trials aiming at optimizing chemotherapy protocols as well as local therapies in a stepwise approach. Most of the new anticancer drugs currently in development are based on targeted therapies, directed to specific targets present only in or on tumor cells, like growth factor receptors, mechanisms involved in proliferation, DNA repair, apoptosis, tumor invasion or angiogenesis. Concerning bone marrow transplantation also, new strategic approaches are in advanced development. They aim at reducing treatment induced toxicity and enhancing efficacy at the same time. This short paper would like to point out these new technologies, which should be known by the general practitioner.

Régulation du processus métastatique tumoral par l'oxyde nitrique (NO), le TGF-β el les cellules de l'hôte

RESUME Nous avons étudié le rôle de deux molécules, le Transfon-ning Growth Factor (TGF-β) et l'oxyde nitrique (NO), dans le processus métastatique. Deux clones tumoraux ont été sélectionnés à partir d'un carcinome du côlon pour leur différence de potentiel tumorigénique dans des rats syngéniques. La croissance tumorale du clone progressif PROb a été corrélée à sa capacité à sécréter le TGF-β actif Cependant, la transfection du clone régressif REGb, sécrétant du TGF-β latent, par une vecteur codant pour le TGF-β bio-actif n'a pas permis d'induire le développement tumoral. Les deux clones tumoraux présentent des activités des protéases MMP-2, APN et DPPIV identiques et qui ne semblent pas modifiées par le TGF-β. L'interaction des cellules tumorales avec l'endothélium et l'activité de la NO synthase (iNOS) responsable de la synthèse de NO sont impliqués dans la progression de nombreux cancers. Le clone PROb, mais pas le clone REGb, inhibe l'activation de la iNOS des cellules endothéliales par sa sécrétion de TGF-β actif Les deux clones montrent cependant des propriétés d'adhésion identiques aux cellules endothéliales et sont capables d'inhiber par contact cellulaire direct l'activation de la iNOS endothéliale. Ceci suggère que ces contacts directs pourraient créer un micro-environnement favorable à la conversion du TGF-β latent en TGF-β actif ou à d'autres interactions moléculaires pouvant réguler l'activation endothéliale. Par ailleurs, les deux clones activent des macrophages du système nerveux central, organe où ils ne forment pas de métastases, mais pas les macrophages circulants, illustrant des mécanismes différentiels et spécifiques dans l'activation de différents types de cellules immunitaires. Afin de mieux comprendre le rôle du NO dans la dissémination métastatique, deux clones cellulaires différant par le taux d'activité de la iNOS ont été sélectionnés à partir de la lignée murine parentale de carcinome du sein EMT-6. Bien que le NO soit un inhibiteur potentiel de la prolifération cellulaire, les deux clones montrent des propriétés prolifératives identiques in vitro. Les cellules EMT-6H qui produisent peu de NO in vitro forment de nombreux nodules tumoraux pulmonaires in vivo corrélés à une mortalité significative des souris syngéniques injectées. Les cellules EMT-6J qui présentent une expression élevée de iNOS et de NO induisent de rares nodules tumoraux pulmonaires et peu de mortalité. Dans ce modèle, l'expression tumorale de NO semble donc défavoriser la croissance tumorale. Les deux clones cellulaires ont des propriétés identiques d'adhésion et de prolifération mesurées in vitro sur des cellules endothéliales primaires isolées de différents organes et in vivo par une colocalisation identique dans les poumons de souris syngéniques 48h après leur injection. Les cellules EMT-6H présentent une activité MMP-2 plus élevée alors que les activités des protéases APN et DPPIV sont identiques dans les deux clones cellulaires. Le TGF-β soluble ainsi que les fibroblastes primaires bloquent la prolifération des deux clones cellulaires. Cependant, l'activation préalable des fibroblastes par du TGF-β restaure partiellement la prolifération du clone EMT-6H mais pas celle du clone EMT-6J. Ces résultats montrent que le rôle de molécules telles que le TGF-β et le NO tumoral dans la progression tumorale doit être considéré dans un contexte d'interactions des cellules tumorales avec les différentes types cellulaires de l'hôte: en particulier, notre travail souligne que les macrophages et les fibroblastes sont déterminants dans la progression métastatique des carcinomes du côlon ou du sein. RESUME DESTINE A UN LARGE PUBLIC Les métastases tumorales, disséminées et intraitables par chirurgie, représentent un problème majeur dans le traitement clinique du cancer. Elles sont dues à des cellules tumorales qui ont migré de leur site tumoral primaire, circulé et survécu dans le système vasculaire de l'hôte, échappé au système immunitaire, adhéré à et survécu sur l'endothélium des vaisseaux, et envahi le tissu sous-jacent où elles ont proliféré. Cette capacité à former des métastases implique de nombreux facteurs dont certains ont été identifiés mais dont le rôle reste controversé dans les différentes études. Nous nous sommes intéressés au rôle de l'oxyde nitrique (NO) et du facteur de croissance et de transformation cellulaire TGF-β. Dans les carcinomes du sein, l'expression des enzymes responsables de la synthèse de NO a été corrélée avec l'invasion tumorale mais aussi avec un pronostic favorable selon les études. Deux clones cellulaires ont été isolés à partir de la tumeur mammaire EMT-6 chez la souris. Le clone EMT-6H sécrète peu de NO et forme de nombreuses tumeurs dans les poumons des souris *entraînant leur décès. Le clone EMT-6J sécrète beaucoup de NO et ne se développe que peu dans les poumons. Dans ce modèle expérimental, le NO semble donc défavoriser la croissance tumorale. L'analyse des interactions avec les cellules de l'hôte rencontrées lors de la formation de métastases pulmonaires a montré que les deux clones cellulaires adhérent et prolifèrent de manière similaire sur les cellules endothéliales tapissant l'intérieur des vaisseaux sanguins. L'arrêt des cellules tumorales dans les poumons ne permet donc pas d'expliquer la différence de croissance tumorale. Cependant, le clone agressif EMT-6H présente une activité élevée d'une protéase (MMP-2) qui lui permettrait par la suite d'envahir le tissu pulmonaire. Par ailleurs, l'activation des fibroblastes du tissu pulmonaire par le TGF-β, une molécule observée dans des conditions inflammatoires, permet au clone agressif EMT-6H de proliférer mais inhibe la croissance du clone EMT-6J. Dans un modèle expérimental de carcinome du côlon, le TGF-β est considéré favorable à la croissance tumorale. Isolées à partir de la même tumeur initiale, deux lignées de cellules ont des comportements opposés lorsqu'elles sont injectées sous la peau des rats. La capacité de la lignée PROb à former des tumeurs a été corrélée à la sécrétion de TGF-β actif L'introduction du gène codant pour le TGF-β actif dans la lignée REGb, qui ne sécrète pas de TGF-β actif et ne forme pas de tumeurs chez le rat, ne restaure pas leur potentiel tumorigénique. Dans ce modèle, l'expression de TGF-β actif ne semble donc pas suffisante à la croissance tumorale. Les interactions avec différents types cellulaires de l'hôte ont été étudiées. Les deux lignées tumorales adhérent de manière similaire sur les cellules endothéliales et sont capables d'inhiber leur activation, un mécanisme qui pourrait participer à la destruction. Les deux lignées activent les cellules immunitaires du système nerveux central, un organe où elles ne forment pas de métastase. Ces résultats suggèrent que la sélection des cellules métastatiques ne s'effectue pas sur l'endothélium des vaisseaux sanguins mais à des étapes ultérieures dans le micro- environnement cellulaire du nouvel organe colonisé. SUMMARY Metastasis results from the migration of tumor cells from their primary tumor, circulation through the bloodstream, attachment to the endothelium, and invasion of the surrounding tissue where they create a microenvironnement favoring their growth. This multistep process implies various cellular interactions and molecules. Among those, we were interested in the role of the Transforming Growth Factor beta (TGF-β) and the nitric oxide (NO). Two cell lines were isolated from a rat colon tumor and assessed for their metastatic potential in vivo. The PROb cell line that expresses active TGF-β formed subcutaneous tumors in rats while the REGb cell line that expresses only latent TGF-β did not. Transfection of REGb cells with a plasmid encoding for the active form of TGF-β failed to restore their metastatic ability. Thus TGF-β secretion is not sufficient to induce colon carcinoma progression. Activities of various proteases such as APN, DPPIV and MMP were similar in both cell lines and were not regulated by TGF-β. Interactions with the endothelium as well as NO synthase activity (iNOS) and local NO concentrations are believed to be crucial steps in cancer metastasis. Coculture of the two clones with endothelial cells inhibited the cytokine-triggered activation of the iNOS enzyme in primary rat endothelial cells but only PROb cells were capable of increasing the expression of IL-6, a protumoral interleukin that may participate in the impairment of the anti-tumoral immune response of the host. Both cell lines exhibited potential to activate microglial cells but not bone marrow-derived macrophages, pointing to a differential regulation of specialized immune cells. To better understand the conflicting role of NO in breast cancer progression, two cell clones were selected from the murine tumorigenic cell line EMT-6 based on their iNOS activity and NO secretion. Although NO has been shown to inhibit cell proliferation, the two cell clones exhibited similar proliferation rates in vitro. The EMT-6H cells expressed little NO and grew actively in the lungs of syngenic mice, leading to their death. Opposite results were observed with the EMT-6J cells. In these in vivo conditions, NO seems to impair tumor growth. Both clones exhibited similar in vitro adhesive properties to primary endothelial cells isolated from various mouse organs and similar localization in the lungs of mice 48 hours after injection. Sustained metalloproteinase MMP-2 activity was detected in the tumorigenic EMT-6H clone, but not in the EMT-6J cells while other proteases such as APN and DPPIV showed no difference. These results suggested that the two clones differed in invasion steps following adhesion to the endothelium and that NO did not participate in previous steps. Consistent with this, both soluble TGF-β and supernatants of cultures of mouse primary lung fibroblasts inhibited the growth of the two clones. However, previous activation of these fibroblasts with TGF-β restored the growth of the tumorigenic EMT-6H cells, but not of EMT-6J cells. Altogether, these results indicate that the role of a given molecule, such as NO or TGF-β, must be considered in a context of interaction of tumor cells with host cells. They further imply that interaction of tumor cells with specialized immune cells and with stromal cells of the colonized organ, rather than with the endothelium, are critical in regulating metastasis.

Basic calcium phosphate crystals induce monocyte/macrophage IL-1(beta) secretion through the NLRP3 inflammasome in vitro.

Basic calcium phosphate (BCP) crystals are associated with severe osteoarthritis and acute periarticular inflammation. Three main forms of BCP crystals have been identified from pathological tissues: octacalcium phosphate, carbonate-substituted apatite, and hydroxyapatite. We investigated the proinflammatory effects of these BCP crystals in vitro with special regard to the involvement of the NLRP3-inflammasome in THP-1 cells, primary human monocytes and macrophages, and mouse bone marrow-derived macrophages (BMDM). THP-1 cells stimulated with BCP crystals produced IL-1β in a dose-dependent manner. Similarly, primary human cells and BMDM from wild-type mice also produced high concentrations of IL-1β after crystal stimulation. THP-1 cells transfected with short hairpin RNA against the components of the NLRP3 inflammasome and mouse BMDM from mice deficient for NLRP3, apoptosis-associated speck-like protein, or caspase-1 did not produce IL-1β after BCP crystal stimulation. BCP crystals induced macrophage apoptosis/necrosis as demonstrated by MTT and flow cytometric analysis. Collectively, these results demonstrate that BCP crystals induce IL-1β secretion through activating the NLRP3 inflammasome. Furthermore, we speculate that IL-1 blockade could be a novel strategy to inhibit BCP-induced inflammation in human disease.

Beta-catenin is dispensable for hematopoiesis and lymphopoiesis.

Beta-catenin-mediated Wnt signaling has been suggested to be critically involved in hematopoietic stem cell maintenance and development of T and B cells in the immune system. Unexpectedly, here we report that inducible Cre-loxP-mediated inactivation of the beta-catenin gene in bone marrow progenitors does not impair their ability to self-renew and reconstitute all hematopoietic lineages (myeloid, erythroid, and lymphoid), even in competitive mixed chimeras. In addition, both thymocyte survival and antigen-induced proliferation of peripheral T cells is beta-catenin independent. In contrast to earlier reports, these data exclude an essential role for beta-catenin during hematopoiesis and lymphopoiesis.

Dynamic regulation of notch 1 and notch 2 surface expression during T cell development and activation revealed by novel monoclonal antibodies.

It is well established that Notch signaling plays a critical role at multiple stages of T cell development and activation. However, detailed analysis of the cellular and molecular events associated with Notch signaling in T cells is hampered by the lack of reagents that can unambiguously measure cell surface Notch receptor expression. Using novel rat mAbs directed against the extracellular domains of Notch1 and Notch2, we find that Notch1 is already highly expressed on common lymphoid precursors in the bone marrow and remains at high levels during intrathymic maturation of CD4(-)CD8(-) thymocytes. Notch1 is progressively down-regulated at the CD4(+)CD8(+) and mature CD4(+) or CD8(+) thymic stages and is expressed at low levels on peripheral T cells. Immunofluorescence staining of thymus cryosections further revealed a localization of Notch1(+)CD25(-) cells adjacent to the thymus capsule. Notch1 was up-regulated on peripheral T cells following activation in vitro with anti-CD3 mAbs or infection in vivo with lymphocytic chorio-meningitis virus or Leishmania major. In contrast to Notch1, Notch2 was expressed at intermediate levels on common lymphoid precursors and CD117(+) early intrathymic subsets, but disappeared completely at subsequent stages of T cell development. However, transient up-regulation of Notch2 was also observed on peripheral T cells following anti-CD3 stimulation. Collectively our novel mAbs reveal a dynamic regulation of Notch1 and Notch2 surface expression during T cell development and activation. Furthermore they provide an important resource for future analysis of Notch receptors in various tissues including the hematopoietic system.

Functional analysis of different haematopoietic stem cell subsets

RÉSUMÉ : Elucider les bases moléculaires et cellulaires du fonctionnement des cellules souches s'avère crucial dans la compréhension de l'organisation cellulaire au sein des tissus et des organes ainsi que pour le développement de nouvelles stratégies thérapeutiques en médecine régénérative et en oncologie. Les cellules souches adultes les mieux connues sont celles responsables de l'hématopoïèse, les cellules souches hématopoïétiques (CSH). Durant ces dernières années, la recherche a porté une attention particulière à l'isolation prospective de CSH dérivées de la moelle osseuse de souris en utilisant des marqueurs de surface cellulaire ainsi que des propriétés fonctionnelles alléguées. Par la suite, la capacité fonctionnelle des CSH a été vérifiée classiquement par leur transplantation intraveineuse dans des souris réceptrices conditionnées et par l'analyse de leur aptitude à reconstituer le système hématopoïétique à long terme. Des études récentes suggèrent que la transplantation des cellules directement dans la moelle osseuse pourrait non seulement aboutir à une prise de greffe plus rapide et plus efficace, mais pourrait même aider à l'identification de cellules qui ont certes des propriétés intrinsèques de CSH, mais qui n'ont pas la capacité de trouver leur niche au sein de la moelle osseuse et ont donc échoué dans les analyses classiques de reconstitution. Dans cette étude, nous comparons à deux niveaux la fonction de différents sous-groupes de cellules souches de la moelle osseuse, définis par leur phénotype de surface cellulaire. Premièrement, nous étudions leur capacité à reconstituer des souris létalement irradiées après injection soit intraveineuse soit intrafémorale. Deuxièmement, par analyse cytométrique de flux à 8 couleurs, nous comparons leur activité relative de « side population » (SP) par exclusion du colorant fluorescent Hoechst 33342. Nos résultats préliminaires renforcent en effet l'idée que la transplantation intrafémorale aboutit à une greffe plus rapide et plus efficace. Par contre, en utilisant cette approche, nous n'arrivons pas à identifier des cellules capables de prendre greffe spécifiquement quand elles sont injectées en intrafémorale. Finalement, bien qu'une confirmation in vivo soit encore nécessaire, nous suggérons sur la base de nos analyses cytométriques de flux, que les cellules SP Sca1t~és éie~~ CD48t~és bas sont très enrichies en CSH. Ceci permettrait l'isolation ex vivo de CSH de la moelle osseuse de souris par une stratégie à la fois nouvelle et simple. SUMMARY : Elucidating the molecular and cellular bases of stem cell function is crucial for the understanding of cellular organisation within tissues and organs as well as for the development of new therapeutic strategies in regenerative medicine and oncology. The best-known adult stem cells are those responsible for haematopoiesis, the haematopoietic stem cells (HSCs). In recent years, much effort has been put into the prospective isolation of mouse bone marrow (BM)-derived HSCs using cell-surface markers and alleged functional properties. Upon isolation, the functional capacity of putative HSCs has been classically assessed by intravenous transplantation into conditioned recipient mice and analysis of their ability to reconstitute the haematopoietic system at long-term. It has recently been suggested that transplanting the cells directly into the BM might not only result in more rapid and more effective engraftment, but even help to identify cells that have intrinsic HSC properties but lack the ability to home to their BM niche and have thus failed to succeed in classical reconstitution assays. In this study, we compare the function of different BM cell subsets, as defined by their cell surface phenotype, on two levels. Firstly, we assess their ability to reconstitute lethally irradiated mice, when injected either intravenously or intrafemorally. Secondly, using 8-colour flow cytometric analysis, we compare their relative side population (SP) activity by exclusion of the fluorescent dye Hoechst 33342. Our preliminary results indeed reinforce the idea that intrafemoral transplantation results in faster and more effective engraftment, however, using this approach, we are unable to identify cells that are capable of engrafting specifically when injected intrafemorally. Finally, although in vivo confirmation is still required, we propose, based on the results of our flow cytometric analyses, that SP Scat Very h'9h CD48Very'°W cells should be highly enriched for HSCs. This would allow for a simple new strategy for the isolation of mouse BM HSCs ex vivo.