The Natural Cell-Penetrating Peptide Crotamine Targets Tumor Tissue in Vivo and Triggers a Lethal Calcium-Dependent Pathway in Cultured Cells

Our goal was to demonstrate the in vivo tumor specific accumulation of crotamine, a natural peptide from the venom of the South American rattlesnake Crotalus durissus terrificus, which has been characterized by our group as a cell penetrating peptide with a high specificity for actively proliferating cells and with a concentration-dependent cytotoxic effect. Crotamine cytotoxicity has been shown to be dependent on the disruption of lysosomes and subsequent activation of intracellular proteases. In this work, we show that the cytotoxic effect of crotamine also involves rapid intracellular calcium release and loss of mitochondrial membrane potential as observed in real time by confocal microscopy. The intracellular calcium overload induced by crotamine was almost completely blocked by thapsigargin. Microfluorimetry assays confirmed the importance of internal organelles, such as lysosomes and the endoplasmic reticulum, as contributors for the intracellular calcium increase, as well as the extracellular medium. Finally, we demonstrate here that crotamine injected intraperitoneally can efficiently target remote subcutaneous tumors engrafted in nude mice, as demonstrated by a noninvasive optical imaging procedure that permits in vivo real-time monitoring of crotamine uptake into tumor tissue. Taken together, our data indicate that the cytotoxic peptide crotamine can be used potentially for a dual purpose: to target and detect growing tumor tissues and to selectively trigger tumor cell death.

The Cytoplasmic Zinc Finger Protein ZPR1 Accumulates in the Nucleolus of Proliferating Cells

The zinc finger protein ZPR1 translocates from the cytoplasm to the nucleus after treatment of cells with mitogens. The function of nuclear ZPR1 has not been defined. Here we demonstrate that ZPR1 accumulates in the nucleolus of proliferating cells. The role of ZPR1 was examined using a gene disruption strategy. Cells lacking ZPR1 are not viable. Biochemical analysis demonstrated that the loss of ZPR1 caused disruption of nucleolar function, including preribosomal RNA expression. These data establish ZPR1 as an essential protein that is required for normal nucleolar function in proliferating cells.

Influence of triamcinolone intravitreal injection on retinochoroidal healing processes.

To analyze the effects of triamcinolone intravitreal injection on the wound healing processes after argon laser retinal photocoagulation, wild type C57BL/6J mice, 8-12 weeks old underwent a standard argon laser photocoagulation protocol. After pentobarbital anesthesia and pupil dilatation, argon laser lesions were induced (50microm, 400mW, 0.05s). Two photocoagulation impacts created two disc diameters from the optic nerve in both eyes. The photocoagulated mice were divided into four groups: Group I (n=12), photocoagulation controls, did not receive any intravitreous injection. Group II (n=12), received an intravitreous injection of 1microl of balanced salt solution (BSS). Group III (n=12), received an intravitreous injection of 1microl containing 15microg of triamcinolone acetonide (TAAC) in BSS. Two mice from each of these three groups were sacrificed at 1, 3, 7, 14 days and 2 and 4 months after photocoagulation. Group IV (n=10) received 1.5, 3, 7.5, 15, or 30microg of TAAC and were all sacrificed on day 14. The enucleated eyes were subjected to systematic analysis of the cellular remodeling processes taking place within the laser lesion and its vicinity. To this purpose, specific antibodies against GFAP, von Willebrand factor, F4/80 and KI67 were used for the detection of astrocytes, activated Müller cells, vascular endothelial cells, infiltrating inflammatory cells and actively proliferating cells. TUNEL reaction was also carried out along with nuclear DAPI staining. Temporal and spatial observations of the created photocoagulation lesions demonstrate that 24h following the argon laser beam, a localized and well-delineated affection of the RPE cells and choroid is observed in mice in Groups I and II. The inner retinal layers in these mice eyes are preserved while TUNEL positive (apoptotic) cells are observed at the retinal outer nuclear layer level. At this stage, intense staining with GFAP is associated with activated retinal astrocytes and Müller cells throughout the laser path. From day 3 after photocoagulation, dilated new choroidal capillaries are detected on the edges of the laser lesion. These processes are accompanied by infiltration of inflammatory cells and the presence of proliferating cells within the lesion site. Mice in Group III treated with 15microg/mul of triamcinolone showed a decreased number of infiltrating inflammatory cells and proliferating cells, which was not statistically significant compared to uninjected laser treated controls. The development of new choroidal capillaries on the edges of the laser lesion was also inhibited during the first 2 months after photocoagulation. However, on month 4 the growth of new vessels was observed in these mice treated with TAAC. Mice of Group IV did not show any development of new capillaries even with small doses. After argon laser photocoagulation of the mouse eye, intravitreal injection of triamcinolone markedly influenced the retina and choroid remodeling and healing processes. Triamcinolone is a powerful inhibitor of the formation of neovessels in this model. However, this inhibition is transient. These observations should provide a practical insight for the mode of TAAC use in patients with wet AMD.

DNA-Interactive Properties of Crotamine, a Cell-Penetrating Polypeptide and a Potential Drug Carrier

Crotamine, a 42-residue polypeptide derived from the venom of the South American rattlesnake Crotalus durissus terrificus, has been shown to be a cell-penetrating protein that targets chromosomes, carries plasmid DNA into cells, and shows specificity for actively proliferating cells. Given this potential role as a nucleic acid-delivery vector, we have studied in detail the binding of crotamine to single- and double-stranded DNAs of different lengths and base compositions over a range of ionic conditions. Agarose gel electrophoresis and ultraviolet spectrophotometry analysis indicate that complexes of crotamine with long-chain DNAs readily aggregate and precipitate at low ionic strength. This aggregation, which may be important for cellular uptake of DNA, becomes less likely with shorter chain length. 25-mer oligonucleotides do not show any evidence of such aggregation, permitting the determination of affinities and size via fluorescence quenching experiments. The polypeptide binds non-cooperatively to DNA, covering about 5 nucleotide residues when it binds to single (ss) or (ds) double stranded molecules. The affinities of the protein for ss-vs. ds-DNA are comparable, and inversely proportional to salt levels. Analysis of the dependence of affinity on [NaCl] indicates that there are a maximum of,3 ionic interactions between the protein and DNA, with some of the binding affinity attributable to non-ionic interactions. Inspection of the three-dimensional structure of the protein suggests that residues 31 to 35, Arg-Trp-Arg-Trp-Lys, could serve as a potential DNA-binding site. A hexapeptide containing this sequence displayed a lower DNA binding affinity and salt dependence as compared to the full-length protein, likely indicative of a more suitable 3D structure and the presence of accessory binding sites in the native crotamine. Taken together, the data presented here describing crotamine-DNA interactions may lend support to the design of more effective nucleic acid drug delivery vehicles which take advantage of crotamine as a carrier with specificity for actively proliferating cells. Citation: Chen P-C, Hayashi MAF, Oliveira EB, Karpel RL (2012) DNA-Interactive Properties of Crotamine, a Cell-Penetrating Polypeptide and a Potential Drug Carrier. PLoS ONE 7(11): e48913. doi:10.1371/journal.pone.0048913

Specificità, efficacia e risposte adattative mediate da mutazioni dell'oncosoppressore TP53 degli inibitori del Complesso I mitocondriale nella terapia oncologica personalizzata: dai meccanismi molecolari allo sviluppo di modelli preclinici avanzati

L'inibizione del complesso respiratorio I (CI) è una strategia antitumorale emergente, sebbene la specificità e l’efficacia di nuovi farmaci restino poco investigate. La generazione di modelli cellulari tumorali nulli per il CI rivela la specificità di EVP 4593 e BAY 872243 nell’indurre gli effetti antiproliferativi non associati all’apoptosi, selettivamente via CI, riducendo eventuali effetti collaterali. Studi preliminari in vivo evidenziano un rallentamento della crescita tumorale negli animali trattati con EVP 4593, il quale emerge come l’inibitore più potente. Per il suo ruolo nella riprogrammazione metabolica, e la sua elevata frequenza di mutazioni nelle neoplasie umane, sono stati investigati i potenziali meccanismi di adattamento alla terapia anti-CI sulla base dello stato mutazionale di TP53. L’auxotrofia da aspartato, un hallmark metabolico delle cellule tumorali con un danno al CI, causa un blocco della sintesi proteica mTORC1-dipendente nelle linee cellulari con una p53 mutata o nulla, inducendo un collasso metabolico. Viceversa, l'attivazione del sensore energetico AMPK promuove un recupero parziale della sintesi di aspartato in linee cellulari con la forma wild type di P53, che è in grado di sostenere una migliore anaplerosi attraverso SCO2, fattore di assemblaggio del complesso respiratorio IV. Al fine di traslare questi risultati in un modello preclinico, si è ottimizzato l’ottenimento di colture di tumori umani espiantati tramite il bioreattore U-CUP. Il modello scelto è stato quello di carcinoma sieroso ad alto grado dell’ovaio (HGSOC), a partire da tessuto congelato, per l’elevata frequenza di mutazioni driver in TP53. I tessuti congelati preservano l'eterogeneità delle componenti cellulari del tessuto di origine e sono caratterizzati da cellule in attiva proliferazione senza attivazione di apoptosi. Dati preliminari mostrano un trend di riduzione dell’area tumorale nei tessuti trattati con EVP 4593 e supportano l’utilizzo del modello preclinico nello studio di nuovi inibitori del CI sfruttando materiale primario di pazienti oncologici.

Phenotypic and molecular characterization of proliferating and differentiated GnRH-expressing GnV-3 cells.

GnRH neurons provide the primary driving force upon the neuroendocrine reproductive axis. Here we used GnV-3 cells, a model of conditionally immortalized GnRH-expressing neurons, to perform an analysis of cell cycle and compare the gene expression profile of proliferating cells with differentiated cells. In the proliferation medium, 45 ± 1.5% of GnV-3 cells are in S-phase by FACS analysis. In the differentiation medium, only 9 ± 0.9% of them are in S-phase, and they acquire the characteristic bipolar shape displayed by preoptic GnRH neurons in vivo. In addition, GnV-3 cells in the differentiated state exhibit electrophysiological properties characteristic of neurons. Transcriptomic analysis identified up-regulation of 1931 genes and down-regulation of 1270 genes in cells grown in the differentiation medium compared to cells in the proliferation medium. Subsequent gene ontology study indicated that genes over-expressed in proliferating GnV-3 cells were mainly involved in cell cycle regulations, whereas genes over-expressed in differentiated cells were mainly involved in processes of differentiation, neurogenesis and neuronal morphogenesis. Taken together, these data demonstrate the occurrence of morphological and physiological changes in GnV-3 cells between the proliferating and the differentiated state. Moreover, the genes differentially regulated between these two different states are providing novel pathways potentially important for a better understanding of the physiology of mature GnRH neurons.

Human cardiospheres as a source of multipotent stem and progenitor cells.

Cardiospheres (CSs) are self-assembling multicellular clusters from the cellular outgrowth from cardiac explants cultured in nonadhesive substrates. They contain a core of primitive, proliferating cells, and an outer layer of mesenchymal/stromal cells and differentiating cells that express cardiomyocyte proteins and connexin 43. Because CSs contain both primitive cells and committed progenitors for the three major cell types present in the heart, that is, cardiomyocytes, endothelial cells, and smooth muscle cells, and because they are derived from percutaneous endomyocardial biopsies, they represent an attractive cell source for cardiac regeneration. In preclinical studies, CS-derived cells (CDCs) delivered to infarcted hearts resulted in improved cardiac function. CDCs have been tested safely in an initial phase-1 clinical trial in patients after myocardial infarction. Whether or not CDCs are superior to purified populations, for example, c-kit(+) cardiac stem cells, or to gene therapy approaches for cardiac regeneration remains to be evaluated.

Establishment of models to study mouse and human retinogenesis "in vitro" : isolation and characterization of retinal stem cells

SUMMARY IN FRENCH Les cellules souches sont des cellules indifférenciées capables a) de proliférer, b) de s'auto¬renouveller, c) de produire des cellules différenciées, postmitotiques et fonctionnelles (multipotencialité), et d) de régénérer le tissu après des lésions. Par exemple, les cellules de souches hematopoiétiques, situées dans la moelle osseuse, peuvent s'amplifier, se diviser et produire diverses cellules différenciées au cours de la vie, les cellules souches restant dans la moelle osseuse et consentant leur propriété. Les cellules souches intestinales, situées dans la crypte des microvillosités peuvent également régénérer tout l'intestin au cours de la vie. La rétine se compose de six classes de neurones et d'un type de cellule gliale. Tous ces types de cellules sont produits par un progéniteur rétinien. Le pic de production des photorécepteurs se situe autour des premiers jours postnatals chez la souris. A cette période la rétine contient les cellules hautement prolifératives. Dans cette étude, nous avons voulu analyser le phénotype de ces cellules et leur potentiel en tant que cellules souches ou progénitrices. Nous nous sommes également concentrés sur l'effet de certains facteurs épigéniques sur leur destin cellulaire. Nous avons observé que toutes les cellules prolifératives isolées à partir de neurorétines postnatales de souris expriment le marqueur de glie radiaire RC2, ainsi que des facteurs de transcription habituellement trouvés dans la glie radiaire (Mash1, Pax6), et répondent aux critères des cellules souches : une capacité élevée d'expansion, un état indifférencié, la multipotencialité (démontrée par analyse clonale). Nous avons étudié la différentiation des cellules dans différents milieux de culture. En l'absence de sérum, l'EGF induit l'expression de la β-tubulin-III, un marqueur neuronal, et l'acquisition d'une morphologie neuronale, ceci dans 15% des cellules présentes. Nous avons également analysé la prolifération de cellules. Seulement 20% des cellules incorporent le bromodéoxyuridine (BrdU) qui est un marqueur de division cellulaire. Ceci démontre que l'EGF induit la formation des neurones sans une progression massive du cycle cellulaire. Par ailleurs, une stimulation de 2h d'EGF est suffisante pour induire la différentiation neuronale. Certains des neurones formés sont des cellules ganglionnaires rétiniennes (GR), comme l'indique l'expression de marqueurs de cellules ganglionnaires (Ath5, Brn3b et mélanopsine), et dans de rare cas d'autres neurones rétiniens ont été observés (photorécepteurs (PR) et cellules bipolaires). Nous avons confirmé que les cellules souches rétiniennes tardives n'étaient pas restreintes au cours du temps et qu'elles conservent leur multipotencialité en étant capables de générer des neurones dits précoces (GR) ou tardifs (PR). Nos résultats prouvent que l'EGF est non seulement un facteur contrôlant le développement glial, comme précédemment démontré, mais également un facteur efficace de différentiation pour les neurones rétiniens, du moins in vitro. D'autre part, nous avons voulu établir si l'oeil adulte humain contient des cellules souches rétiniennes (CSRs). L'oeil de certains poissons ou amphibiens continue de croître pendant l'âge adulte du fait de l'activité persistante des cellules souches rétiniennes. Chez les poissons, le CSRs se situe dans la marge ciliaire (CM) à la périphérie de la rétine. Bien que l'oeil des mammifères ne se développe plus pendant la vie d'adulte, plusieurs groupes ont prouvé que l'oeil de mammifères adultes contient des cellules souches rétiniennes également dans la marge ciliaire plus précisément dans l'épithélium pigmenté et non dans la neurorétine. Ces CSRs répondent à certains critères des cellules souches. Nous avons identifié et caractérisé les cellules souches rétiniennes résidant dans l'oeil adulte humain. Nous avons prouvé qu'elles partagent les mêmes propriétés que leurs homologues chez les rongeurs c.-à-d. auto-renouvellement, amplification, et différenciation en neurones rétiniens in vitro et in vivo (démontré par immunocoloration et microarray). D'autre part, ces cellules peuvent être considérablement amplifiées, tout en conservant leur potentiel de cellules souches, comme indiqué par l'analyse de leur profil d'expression génique (microarray). Elles expriment également des gènes communs à diverses cellules souches: nucleostemin, nestin, Brni1, Notch2, ABCG2, c-kit et son ligand, aussi bien que cyclin D3 qui agit en aval de c-kit. Nous avons pu montré que Bmi1et Oct4 sont nécessaires pour la prolifération des CSRs confortant leur propriété de cellules souches. Nos données indiquent que la neurorétine postnatale chez la souris et l'épithélium pigmenté de la marge ciliaire chez l'humain adulte contiennent les cellules souches rétiniennes. En outre, nous avons développé un système qui permet d'amplifier et de cultiver facilement les CSRs. Ce modèle permet de disséquer les mécanismes impliqués lors de la retinogenèse. Par exemple, ce système peut être employé pour l'étude des substances ou des facteurs impliqués, par exemple, dans la survie ou dans la génération des cellules rétiniennes. Il peut également aider à disséquer la fonction de gènes ou les facteurs impliqués dans la restriction ou la spécification du destin cellulaire. En outre, dans les pays occidentaux, la rétinite pigmentaire (RP) touche 1 individu sur 3500 et la dégénérescence maculaire liée à l'âge (DMLA) affecte 1 % à 3% de la population âgée de plus de 60 ans. La génération in vitro de cellules rétiniennes est aussi un outil prometteur pour fournir une source illimitée de cellules pour l'étude de transplantation cellulaire pour la rétine. SUMMARY IN ENGLISH Stem cells are defined as undifferentiated cells capable of a) proliferation, b) self maintenance (self-renewability), c) production of many differentiated functional postmitotic cells (multipotency), and d) regenerating tissue after injury. For instance, hematopoietic stem cells, located in bone marrow, can expand, divide and generate differentiated cells into the diverse lineages throughout life, the stem cells conserving their status. In the villi crypt, the intestinal stem cells are also able to regenerate the intestine during their life time. The retina is composed of six classes of neurons and one glial cell. All these cell types are produced by the retinal progenitor cell. The peak of photoreceptor production is reached around the first postnatal days in rodents. Thus, at this stage the retina contains highly proliferative cells. In our research, we analyzed the phenotype of these cells and their potential as possible progenitor or stem cells. We also focused on the effect of epigenic factor(s) and cell fate determination. All the proliferating cells isolated from mice postnatal neuroretina harbored the radial glia marker RC2, expressed transcription factors usually found in radial glia (Mash 1, Pax6), and met the criteria of stem cells: high capacity of expansion, maintenance of an undifferentiated state, and multipotency demonstrated by clonal analysis. We analyzed the differentiation seven days after the transfer of the cells in different culture media. In the absence of serum, EGF led to the expression of the neuronal marker β-tubulin-III, and the acquisition of neuronal morphology in 15% of the cells. Analysis of cell proliferation by bromodeoxyuridine incorporation revealed that EGF mainly induced the formation of neurons without stimulating massively cell cycle progression. Moreover, a pulse of 2h EGF stimulation was sufficient to induce neuronal differentiation. Some neurons were committed to the retinal ganglion cell (RGC) phenotype, as revealed by the expression of retinal ganglion markers (Ath5, Brn3b and melanopsin), and in few cases to other retinal phenotypes (photoreceptors (PRs) and bipolar cells). We confirmed that the late RSCs were not restricted over-time and conserved multipotentcy characteristics by generating retinal phenotypes that usually appear at early (RGC) or late (PRs) developmental stages. Our results show that EGF is not only a factor controlling glial development, as previously shown, but also a potent differentiation factor for retinal neurons, at least in vitro. On the other hand, we wanted to find out if the adult human eye contains retina stem cells. The eye of some fishes and amphibians continues to grow during adulthood due to the persistent activity of retinal stem cells (RSCs). In fish, the RSCs are located in the ciliary margin zone (CMZ) at the periphery of the retina. Although, the adult mammalian eye does not grow during adult life, several groups have shown that the adult mouse eye contains retinal stem cells in the homologous zone (i.e. the ciliary margin), in the pigmented epithelium and not in the neuroretina. These RSCs meet some criteria of stem cells. We identified and characterized the human retinal stem cells. We showed that they posses the same features as their rodent counterpart i.e. they self-renew, expand and differentiate into retinal neurons in vitro and in vivo (indicated by immunostaining and microarray analysis). Moreover, they can be greatly expanded while conserving their sternness potential as revealed by the gene expression profile analysis (microarray approach). They also expressed genes common to various stem cells: nucleostemin, nestin, Bmil , Notch2, ABCG2, c-kit and its ligand, as well as cyclin D3 which acts downstream of c-kit. Furthermore, Bmil and Oct-4 were required for RSC proliferation reinforcing their stem cell identity. Our data indicate that the mice postnatal neuroretina and the adult pigmented epithelium of adult human ciliary margin contain retinal stem cells. We developed a system to easily expand and culture RSCs that can be used to investigate the retinogenesis. For example, it can help to screen drugs or factors involved, for instance, in the survival or generation of retinal cells. This could help to dissect genes or factors involved in the restriction or specification of retinal cell fate. In Western countries, retinitis pigmentosa (RP) affects 1 out of 3'500 individuals and age-related macula degeneration (AMD) strikes 1 % to 3% of the population over 60. In vitro generation of retinal cells is thus a promising tool to provide an unlimited cell source for cellular transplantation studies in the retina.

The biology of cancer stem cells : part 1: nuclear translocation of CD44 : part 2: IMP2 in glioblastoma cancer stem cells

SummaryCancer stem cells (CSC) are poorly differentiated, slowly proliferating cells, with high tumorigenic potential. Some of these cells, as it has been shown in leukemia, evade chemo- and radiotherapy and recapitulate the tumor composed of CSC and their highly proliferative progeny. Therefore, understanding the molecular biology of those cells is crucial for improvement of currently used anti-cancer therapies.This work is composed of two CSC-related projects. The first deals with CD44, a frequently used marker of CSC; the second involves Imp2 and its role in CSC bioenergetics. PART 1. CD44 is a multifunctional transmembrane protein involved in migration, homing, adhesion, proliferation and survival. It is overexpressed in many cancers and its levels are correlated with poor prognosis. CD44 is also highly expressed by CSC and in many malignancies it is used for CSC isolation.In the present work full-lenght CD44 nuclear localization was studied, including the mechanism of nuclear translocation and its functional role in the nucleus. Full-length CD44 can be found in nuclei of various cell types, regardless of their tumorigenic potential. For nuclear localization, CD44 needs to be first inserted into the cell membrane, from which it is transported via the endocytic pathway. Upon binding to transportinl it is translocated to the nucleus. The nuclear localization signal recognized by transportinl has been determined as the first 20 amino acids of the membrane proximal intracellular domain. Nuclear export of CD44 is facilitated by exportin Crml. Investigation of the function of nuclear CD44 revealed its implication in de novo RNA synthesis.PART 2. Glioblastoma multiforme is the most aggressive and most frequent brain malignancy. It was one of the first solid tumors from which CSC have been isolated. Based on the similarity between GBM CSC and normal stem cells expression of an oncofetal mRNA binding protein Imp2 has been investigated.Imp2 is absent in normal brain as well as in low grade gliomas, but is expressed in over 75% GBM cases and its expression is higher in CSC compared to their more differentiated counterparts. Analysis of mRNA transcripts bound by Imp2 and its protein interactors revealed that in GBM CSC Imp2 may be implicated in mitochondrial metabolism. Indeed, shRNA mediated silencing of protein expression led to decreased mitochondrial activity, decreased oxygen consumption and decreased activity of respiratory chain protein complex I. Moreover, lack of Imp2 severely affected self-renewal and tumorigenicity of GBM CSC. Experimental evidence suggest that GBM CSC depend on mitochondrial oxidative phosphorylation as an energy producing pathway and that Imp2 is a novel regulator of this pathway.RésuméLes cellules cancéreuses souches sont des cellules peu différentiées, à proliferation lente et hautement tumorigénique. Ces cellules sont radio-chimio résistantes et sont capable reformer la tumeur dans sont intégralité, reproduisant l'hétérogénéité cellulaire présent dans la tumeur d'origine. Pour améliorer les therapies antitumorales actuelles il est crucial de comprendre les mécanismes moléculaires qui caractérisent cette sous-population de cellules hautement malignes.Ce travail de thèse se compose de deux projets s'articulant autour du même axe :Le CD44 est une protéine multifonctionnelle et transmembranaire très souvent utilisée comme marqueur de cellules souches tumorales dans différents cancers. Elle est impliquée dans la migration, l'adhésion, la prolifération et la survie des cellules. Lors de ce travail de recherche, nous nous sommes intéressés à la localisation cellulaire du CD44, ainsi qu'aux mécanismes permettant sa translocation nucléaire. En effet, bien que principalement décrit comme un récepteur de surface transmembranaire, le CD44 sous sa forme entière, non clivée en peptides, peut également être observé à l'intérieur du noyau de diverses cellules, quel que soit leur potentiel tumorigénique. Pour passer ainsi d'un compartiment cellulaire à un autre, le CD44 doit d'abord être inséré dans la membrane plasmique, d'où il est transporté par endocytose jusqu'à l'intérieur du cytoplasme. La transportai permet ensuite la translocation nucléaire du CD44 via une « séquence signal » contenue dans les 20 acides aminés du domaine cytoplasmique qui bordent la membrane. A l'inverse, le CD44 est exporté du noyau grâce à l'exportin Crml. En plus des mécanismes décrits ci-dessus, cette étude a également mis en évidence l'implication du CD44 dans la synthèse des ARN, d'où sa présence dans le noyau.Le glioblastome est la plus maligne et la plus fréquente des tumeurs cérébrales. Dans ce second projet de recherche, le rôle de IMP2 dans les cellules souches tumorales de glioblastomes a été étudié. La présence de cette protéine oncofoetale a d'abord été mise en évidence dans 75% des cas les plus agressifs des gliomes (grade IV, appelés glioblastomes), tandis qu'elle n'est pas exprimée dans les grades I à III de ces tumeurs, ni dans le cerveau sain. De plus, IMP2 est apparue comme étant davantage exprimée dans les cellules souches tumorales que dans les cellules déjà différenciées. La baisse de l'expression de IMP2 au moyen de shRNA a résulté en une diminution de l'activité mitochondriale, en une réduction de la consommation d'oxygène ainsi qu'en une baisse de l'activité du complexe respiratoire I.L'inhibition de IMP2 a également affecté la capacité de renouvellement de la population des cellules souches tumorales ainsi que leur aptitude à former des tumeurs.Lors de ce travail de thèse, une nouvelle fonction d'un marqueur de cellules souches tumorales a été mise en évidence, ainsi qu'un lien important entre la bioénergétique de ces cellules et l'expression d'une protéine oncofoetale.

Isolation of cardiovascular precursor cells from the human fetal heart.

Weakening of cardiac function in patients with heart failure results from a loss of cardiomyocytes in the damaged heart. Cell replacement therapies as a way to induce myocardial regeneration in humans could represent attractive alternatives to classical drug-based approaches. However, a suitable source of precursor cells, which could produce a functional myocardium after transplantation, remains to be identified. In the present study, we isolated cardiovascular precursor cells from ventricles of human fetal hearts at 12 weeks of gestation. These cells expressed Nkx2.5 but not late cardiac markers such as α-actinin and troponin I. In addition, proliferating cells expressed the mesenchymal stem cell markers CD73, CD90, and CD105. Evidence for functional cardiogenic differentiation in vitro was demonstrated by the upregulation of cardiac gene expression as well as the appearance of cells with organized sarcomeric structures. Importantly, differentiated cells presented spontaneous and triggered calcium signals. Differentiation into smooth muscle cells was also detected. In contrast, precursor cells did not produce endothelial cells. The engraftment and differentiation capacity of green fluorescent protein (GFP)-labeled cardiac precursor cells were then tested in vivo after transfer into the heart of immunodeficient severe combined immunodeficient mice. Engrafted human cells were readily detected in the mouse myocardium. These cells retained their cardiac commitment and differentiated into α-actinin-positive cardiomyocytes. Expression of connexin-43 at the interface between GFP-labeled and endogenous cardiomyocytes indicated that precursor-derived cells connected to the mouse myocardium. Together, these results suggest that human ventricular nonmyocyte cells isolated from fetal hearts represent a suitable source of precursors for cell replacement therapies.

Compensatory embryonic response to allele-specific inactivation of the murine X-linked gene Hcfc1.

Early in female mammalian embryonic development, cells randomly inactivate one of the two X chromosomes to achieve overall equal inactivation of parental X-linked alleles. Hcfc1 is a highly conserved X-linked mouse gene that encodes HCF-1 - a transcriptional co-regulator implicated in cell proliferation in tissue culture cells. By generating a Cre-recombinase inducible Hcfc1 knock-out (Hcfc1(lox)) allele in mice, we have probed the role of HCF-1 in actively proliferating embryonic cells and in cell-cycle re-entry of resting differentiated adult cells using a liver regeneration model. HCF-1 function is required for both extraembryonic and embryonic development. In heterozygous Hcfc1(lox/+) female embryos, however, embryonic epiblast-specific Cre-induced Hcfc1 deletion (creating an Hcfc1(epiKO) allele) around E5.5 is well tolerated; it leads to a mixture of HCF-1-positive and -negative epiblast cells owing to random X-chromosome inactivation of the wild-type or Hcfc1(epiKO) mutant allele. At E6.5 and E7.5, both HCF-1-positive and -negative epiblast cells proliferate, but gradually by E8.5, HCF-1-negative cells disappear owing to cell-cycle exit and apoptosis. Although generating a temporary developmental retardation, the loss of HCF-1-negative cells is tolerated, leading to viable heterozygous offspring with 100% skewed inactivation of the X-linked Hcfc1(epiKO) allele. In resting adult liver cells, the requirement for HCF-1 in cell proliferation was more evident as hepatocytes lacking HCF-1 fail to re-enter the cell cycle and thus to proliferate during liver regeneration. The survival of the heterozygous Hcfc1(epiKO/+) female embryos, even with half the cells genetically compromised, illustrates the developmental plasticity of the post-implantation mouse embryo - in this instance, permitting survival of females heterozygous for an X-linked embryonic lethal allele.

Étude de Necdin par un modèle de carcinogénèse lié à l’antigène grand T du Virus du polyome

Les virus sont utilisés depuis longtemps dans la recherche sur le cancer et ont grandement contribué à l’avancement des connaissances de même qu’à l’établissement de préceptes importants encore valables aujourd’hui dans le domaine. L’un des défis actuels est de mieux définir les étapes menant à la transition d’une cellule normale à une cellule transformée et c’est sur cette problématique que nous nous sommes penchés. Pour ce faire, nous avons tiré profit de l’utilisation de l’antigène grand-T du virus de polyome (PyLT), un virus capable d’induire des tumeurs chez les rongeurs. Cet oncogène viral à lui seul possède des propriétés intéressantes qui suggèrent que, en plus de l’immortalisation, il peut également contribuer aux événements précoces de la carcinogénèse. Ceci repose principalement sur la capacité de PyLT à induire des tumeurs en souris transgéniques et ce, avec une certaine latence ce qui suggère que des événements supplémentaires sont nécessaires. Ainsi, l’utilisation de PyLT dans un modèle de culture cellulaire permet de disséquer les changements qui lui sont attribuables. Dans un premier temps, l’établissement du profil d'expression génique associé à l'expression de PyLT dans un modèle murin nous a permis de sélectionner un bon nombre de gènes, parmi lesquels figurait Necdin. Nous avons choisi d’étudier Necdin plus en détail puisque peu d’attention était accordée à cette protéine dans le domaine du cancer, malgré que différentes données de la littérature lui suggèrent à la fois des fonctions suppresseurs de tumeur et oncogéniques. Nous avons démontré que, malgré sa fonction proposée de suppresseur de croissance, l’expression de Necdin n’est pas incompatible avec la prolifération dans la lignée cellulaire de souris NIH 3T3 et les cellules primaires humaines (IMR90), bien que l’inhibition de son expression par shARN confère un avantage prolifératif. Nous avons confirmé que Necdin est un gène cible de p53 induit par différents agents génotoxiques, toutefois son expression peut également être régulée de façon p53-indépendante. De plus, Necdin agit négativement sur l’arrêt du cycle cellulaire en réponse à l’activation de p53. Ceci suggère que Necdin est impliqué dans une boucle de régulation négative de la voie de p53 et que l’augmentation anormale de l’expression de Necdin pourrait contribuer à la perturbation la voie du suppresseur de tumeur p53. L’activation de p53 permet l’arrêt transitoire du cycle cellulaire en condition de stress, mais est aussi impliquée dans l’établissement d’un arrêt permanent nommé sénescence. La sénescence est un mécanisme de protection contre l’accumulation de mutations qui peut contribuer à l’initiation du cancer. Vu l’intéressante implication de Necdin dans la régulation de l’activité de p53, nous avons transposé les connaissances acquises du modèle murin à un modèle humain, plus adapté pour l’étude de la sénescence. La caractérisation de l’expression de Necdin dans des fibroblastes primaires humains à différents passages montre que les jeunes cellules en prolifération active expriment Necdin et que son niveau diminue avec l’établissement de la sénescence réplicative. Le même phénomène est observé lors de la sénescence prématurée provoquée par l’expression d’un oncogène et par l’exposition aux radiations ionisantes. De plus, dans des conditions normales de prolifération, la modulation de Necdin par des essais de gain et de perte de fonction n’affecte pas la durée de vie des cellules primaires. Toutefois, en condition de stress génotoxique dû à l’exposition aux irradiations, les cellules surexprimant Necdin présentent une radiorésistance accrue de la même façon que lorsque p53 est inactivé directement. Ce résultat en cellules humaines vient appuyer l’effet observé dans les cellules de souris sur l’impact qu’aura le niveau de Necdin sur la réponse de p53 en condition de stress. Un bref survol a été fait pour aborder de quelle façon nos résultats en culture cellulaire pouvaient se traduire dans des modèles de cancer chez l’humain. Nous avons caractérisé l’expression de Necdin dans deux types différents de cancer. D’abord, dans le cancer de l’ovaire, le niveau élevé de Necdin dans les tumeurs à faible potentiel de malignité (LMP) en comparaison aux cancers agressifs de l’ovaire de type séreux suggère que l’expression de Necdin se limite aux cellules de cancer LMP, qui présente généralement un p53 de type sauvage. Son expression est aussi retrouvée dans deux lignées cellulaires du cancer de l’ovaire non-tumorigéniques en xénogreffe de souris, dont l’une possède un p53 fonctionnel. De plus, la caractérisation de Necdin dans les lignées cellulaires du cancer de la prostate suggère une relation entre son expression et la présence de p53 fonctionnel. Dans le cancer de la prostate, tout comme pour le cancer de l’ovaire, Necdin semble être présent dans les lignées représentant un stade moins avancé de la maladie. L’utilisation de l’oncoprotéine virale PyLT nous a permis de révéler des propriétés intéressantes de Necdin. Nous proposons que dans certains contextes, l’expression constitutive de Necdin pourrait contribuer au cancer en retardant une réponse par p53 appropriée et possiblement en participant à l’augmentation de l’instabilité génomique. La fonction potentiellement oncogénique de Necdin quant à sa relation avec p53 que nous avons révélée requiert davantage d’investigation et les cancers caractérisés ici pourraient constituer de bons modèles à cette fin.

Sustained activation of p53 in confluent nucleotide excision repair-deficient cells resistant to ultraviolet-induced apoptosis

p53 activation is one of the main signals after DNA damage, controlling cell cycle arrest, DNA repair and apoptosis. We have previously shown that confluent nucleotide excision repair (NER)-deficient cells are more resistant to apoptosis induced by ultraviolet irradiation (UV). Here, we further investigated the effect of cell confluence on UV-induced apoptosis in normal and NER-deficient (XP-A and XP-C) cells, as well as the effects of treatments with the ATWATR inhibitor caffeine, and the patterns of p53 activation. Strong p53 activation was observed in either proliferating or confluent cells. Caffeine increased apoptosis levels and inhibited p53 activation in proliferating cells, suggesting a protective role for p53. However, in confluent NER-deficient cells no effect of caffeine was observed. Transcription recovery measurements showed decreased recovery in proliferating XPA-deficient cells, but no recovery was observed in confluent cells. The levels of the cyclin/Cdk inhibitor, p21(Waf1/Cip1), correlated well with p53 activation in proliferating cells. Surprisingly, confluent cells also showed similar activation of p21(Waf1/Cip1). These results indicate that reduced apoptosis in confluent cells is associated with the deficiency in DNA damage removal, since this effect is not clearly observed in NER-proficient cells. Moreover, the strong activation of p53 in confluent cells, which barely respond to apoptosis, suggests that this protein, under these conditions, is not linked to UV-induced cell death signaling. (c) 2008 Elsevier B.V. All rights reserved.

Isolation and characterization of Wharton's jelly-derived multipotent mesenchymal stromal cells obtained from bovine umbilical cord and maintained in a defined serum-free three-dimensional system

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Rapid fusion and syncytium formation of heterologous cells upon expression of the FGFRL1 receptor

The fusion of mammalian cells into syncytia is a developmental process that is tightly restricted to a limited subset of cells. Besides gamete and placental trophoblast fusion, only macrophages and myogenic stem cells fuse into multinucleated syncytia. In contrast to viral cell fusion, which is mediated by fusogenic glycoproteins that actively merge membranes, mammalian cell fusion is poorly understood at the molecular level. A variety of mammalian transmembrane proteins, among them many of the immunoglobulin superfamily, have been implicated in cell-cell fusion, but none has been shown to actively fuse cells in vitro. Here we report that the FGFRL1 receptor, which is up-regulated during the differentiation of myoblasts into myotubes, fuses cultured cells into large, multinucleated syncytia. We used luciferase and GFP-based reporter assays to confirm cytoplasmic mixing and to identify the fusion inducing domain of FGFRL1. These assays revealed that Ig-like domain III and the transmembrane domain are both necessary and sufficient to rapidly fuse CHO cells into multinucleated syncytia comprising several hundred nuclei. Moreover, FGFRL1 also fused HEK293 and HeLa cells with untransfected CHO cells. Our data show that FGFRL1 is the first mammalian protein that is capable of inducing syncytium formation of heterologous cells in vitro.