The role of Pten in the Hematopoietic System and the Hematopoietic Stem Cells

Résumé Identification, localisation et activation des cellules souches hématopoiétiques dormantes in vivo Les cellules souches somatiques sont présentes dans la majorité des tissus régénératifs comme la peau, l'épithélium intestinal et le système hématopoiétique. A partir d'une seule cellule, elles ont les capacités de produire d'autres cellules souches du même type (auto-renouvellement) et d'engendrer un ensemble défini de cellules progénitrices différenciées qui vont maintenir ou réparer leur tissu hôte. Les cellules souches adultes les mieux caractérisées sont les cellules souches hématopoiétiques (HSC), localisées dans la moelle osseuse. Un des buts de mon travail de doctorat était de caractériser plus en profondeur la localisation des HSCs endogènes in vivo. Pour ce faire, la technique "label retaining assay", se basant sur la division peu fréquentes et sur la dormance des cellules souches, a été utilisée. Après un marquage des souris avec du BrdU (analogue à l'ADN) suivi d'une longue période sans BrdU, les cellules ayant incorporés le marquage ("label retaining cells" LCRs) ont pu être identifiées dans la moelle osseuse. Ces cellules LCRs étaient enrichies 300 fois en cellules de phenotype HSC et, en utilisant de la cytofluorométrie, il a pu être montré qu'environ 15% de toutes les HSCs d'une souris restent dormantes durant plusieures semaines. Ces HSCs dormantes à long terme ne sont probablement pas impliquées dans la maintenance de 'hématopoièse. Par contre, on assiste à l'activation rapide de ces HSCs dormantes lors d'une blessure, comme une ablation myéloide. Elles re-entrent alors en cycle cellulaire et sont essentielles pour une génération rapide des cellules progénitrices et matures qui vont remplacer les cellules perdues. De plus, la détection des LCRs, combinée avec l'utilisation du marqueur de HSCs c-kit, peut être utilisée pour la localisation des HSCs dormantes présentes dans la paroi endostéale de la cavité osseuse. De manière surprenante, les LCRs c-kit+ ont surtout étés trouvées isolées en cellule unique, suggérant que le micro-environement spécifique entourant et maintenant les HSCs, appelé niche, pourrait être très réduit et abriter une seule HSC par niche. Rôles complexes du gène supresseur de tumeur Pten dans le système hématopoiétique La phosphatase PTEN disparaît dans certains cancers héréditaires ou sporadiques humains, comme les gliomes, les cancers de l'utérus ou du sein. Pten inhibe la voie de signalisation de la PI3-kinase et joue un rôle clé dans l'apoptose, la croissance, la prolifération et la migration cellulaire. Notre but était d'étudier le rôle de Pten dans les HSC normale et durant la formation de leucémies. Pour ce faire, nous avons généré un modèle murin dans lequel le gène Pten peut être supprimé dans les cellules hématopoiétiques, incluant les HSCs. Ceci a été possible en croissant l'allèle conditionnelle ptenflox soit avec le transgène MxCre inductible par l'interféron α soit avec le transgène Scl-CreERt inductible par le tamoxifen. Ceci permet la conversion de l'allèle ptenflox en l'allèle nul PtenΔ dans les HSCs et les autres types cellulaires hématopoiétiques. Les souris mutantes Pten développent une splénomégalie massive causée par une expansion dramatiques de toutes les cellules myéloides. De manière interessante, alors que le nombre de HSCs dans la moelle osseuse diminue progressivement, le nombre des HSCs dans la rate augmente de manière proportionnelle. Etrangement, les analyses de cycle cellulaire ont montrés que Pten n'avait que peu ou pas d'effet sur la dormance des HSCs ou sur leur autorenouvellement. En revanche, une augmentation massive du niveau de la cytokine de mobilisation G-CSF a été détéctée dans le serum sanguin, suggérant que la suppression de Pten stimulerait la mobilisation et la migration des HSC de la moelle osseuse vers la rate. Finallement, la transplantation de moelle osseuse délétée en Pten dans des souris immuno-déficientes montre que Pten fonctionnerait comme un suppresseur de tumeur dans le système hématopoiétique car son absence entraîne la formation rapide de leucémies lymphocytaires. Summary Identification, localization and activation of dormant hematopoietic stun cells in vivo Somatic stem cells are present in most self-renewing tissues including the skin, the intestinal epithelium and the hematopoietic system. On a single cell basis they have the capacity to produce more stem cells of the same phenotype (self-renewal) and to give rise to a defined set of mature differentiated progeny, responsible for the maintenance or repair of the host tissue. The best characterized adult stem cell is the hematopoietic stem cell (HSC) located in the bone marrow. One goal of my thesis work was to further characterize the location of endogenous HSCs in vivo. To do this, a technique called "label retaining assay» was used which takes advantage of the fact that stem cells (including HSCs) divide very infrequently and can be dormant for months. After labeling mice with the DNA analogue BrdU followed by a long BrdU free "chase", BrdU "label retaining cells" (CRCs) could be identified in the bone marrow. These CRCs were 300-fold enriched for phenotypic HSCs and by using flow cytometry analysis it could be shown that about 15% of all HSCs in the mouse are dormant for many weeks. Our results suggest that these long-term dormant HSCs are unlikely to be involved in homeostatic maintenance. However they are rapidly activated and reenter the cell cycle in response to injury signals such as myeloid ablation. In addition, detection of LRCs in combination with the HSC marker c-Kit could be used to locate engrafted dormant HSCs close to the endosteal lining of the bone marrow cavities. Most surprisingly, c-Kit+LRCs were found predominantly as single cells suggesting that the specific stem cell maintaining microenvironment, called niche, has limited space and may house only single HSCs. Complex roles of the tumor suppressor gene Pten in the hematopoietic system. The phosphatase PTEN is lost in hereditary and sporadic forms of human cancers, including gliomas, endometrial and breast cancers. Pten inhibits the PI3-kina.se pathway and plays a key role in apoptosis, cell growth, proliferation and migration. Our aim was to study the role of Pten in normal HSCs and during leukemia formation. To do this, we generated a mouse model in which the Pten gene can be deleted in hematopoietic cells including HSCs. This was achieved by crossing the conditional ptenflox allele with either the interferona inducible MxCre or the tamoxifen inducible Scl-CreERT transgene. This allowed the conversion of the ptenflox allele into a pterr' null allele in HSCs and other hematopoietic cell types. As a result Pten mutant mice developed massive splenomegaly due to a dramatic expansion of all myeloid cells. Interestingly, while the number of bone marrow HSCs progressively decreased, the number of HSCs in the spleen increased to a similar extent. Unexpectedly, extensive cell cycle analysis showed that Pten had little or no effect on HSC dormancy or HSC self-renewal. Instead, dramatically increased levels of the mobilizing cytokine G-CSF were detected in the blood serum suggesting that loss-of Pten stimulates mobilization and migration of HSC from the BM to the spleen. Finally, transplantation of Pten deficient BM cells into immuno-compromised mice showed that Pten can function as a tumor suppressor in the hematopoietic system and that its absence leads to the rapid formation of T cell leukemia.

Inducible gene and shRNA expression in resident hematopoietic stem cells in vivo.

Hematopoietic stem cells (HSC) are probably the best understood somatic stem cells and often serve as a paradigm for other stem cells. Nevertheless, most current techniques to genetically manipulate them in vivo are either constitutive and/or induced in settings of hematopoietic stress such as after irradiation. Here, we present a conditional expression system that allows for externally controllable transgenesis and knockdown in resident HSCs, based on a lentiviral vector containing a tet-O sequence and a transgenic mouse line expressing a doxycyclin-regulated tTR-KRAB repressor protein. HSCs harvested from tTR-KRAB mice are transduced with the lentiviral vector containing a cDNA (i.e., Green Fluorescent Protein (GFP)) and/or shRNA (i.e., p53) of interest and then transplanted into lethally irradiated recipients. While the vector is effectively repressed by tTR-KRAB during homing and engraftment, robust GFP/shp53 expression is induced on doxycyclin treatment in HSCs and their progeny. Doxycylin-controllable transcription is maintained on serial transplantation, indicating that repopulating HSCs are stably modified by this approach. In summary, this easy to implement conditional system provides inducible and reversible overexpression or knock down of genes in resident HSCs in vivo using a drug devoid of toxic or activating effects.

Ectopic human telomerase catalytic subunit expression maintains telomere length but is not sufficient for CD8+ T lymphocyte immortalization.

Like most somatic human cells, T lymphocytes have a limited replicative life span. This phenomenon, called senescence, presents a serious barrier to clinical applications that require large numbers of Ag-specific T cells such as adoptive transfer therapy. Ectopic expression of hTERT, the human catalytic subunit of the enzyme telomerase, permits fibroblasts and endothelial cells to avoid senescence and to become immortal. In an attempt to immortalize normal human CD8(+) T lymphocytes, we infected bulk cultures or clones of these cells with a retrovirus transducing an hTERT cDNA clone. More than 90% of transduced cells expressed the transgene, and the cell populations contained high levels of telomerase activity. Measuring the content of total telomere repeats in individual cells (by flowFISH) we found that ectopic hTERT expression reversed the gradual loss of telomeric DNA observed in control populations during long term culture. Telomere length in transduced cells reached the levels observed in freshly isolated normal CD8(+) lymphocytes. Nevertheless, all hTERT-transduced populations stopped to divide at the same time as nontransduced or vector-transduced control cells. When kept in IL-2 the arrested cells remained alive. Our results indicate that hTERT may be required but is not sufficient to immortalize human T lymphocytes.

Cell Adhesion Peptide RGDSP and Laminin Support Proliferation and Migration of Postnatal Retinal Stem Cells in a 3D Hydrogel Culture System

Purpose: Retinal stem cells (RSCs) can be isolated from radial glia population of the newborn mouse retina (Angénieux et al., 2006). These RSCs have great capacity to renew and generate neurons including cells differentiated towards the photoreceptor lineage (Mehri-Soussi et al., 2006). However, our published results showed poor integration and survival rate after cell grafting into the retina. The uncontrollable environment of retina seems to be the problem. To bypass this, we are trying to generate hemi-retinal tissue in vitro that can be used for transplantation. Methods: Expanded RSCs were seeded in a mixture of poly-ethylene-glycol (PEG)-polymer-based hydrogels crosslinked by peptides that also serve as substrates for matrix metalloproteinases. Different doses of crosslinker peptides were tested. Several growth factors were studied to stimulate cell proliferation and differentiation. Results: Cells were trapped in hydrogels and cultured in the presence of FGF2 and EGF. Spherical cell clusters indicating proliferation appeared within several days, but there was no cell migration within the gel. We then added cell adhesion molecules integrin ligand RGDSP, or laminin, or a combination of both, into the gel. Cells grown with laminin showed the best proliferation. Cells grown with RGDSP proliferated a few times and then started to spread out. Cells grown with the combination of RGDSP and laminin showed better proliferation than with RGDSP alone and larger spread-outs than with laminin alone. After stimulations with first FGF2 and EGF, and then only FGF2, some cells showed neuronal morphology after 2 weeks. The neuronal population was assessed by the presence of neuronal marker b-tubulin-III. Glial cells were also present. Further characterizations are undergoing. Conclusions: RSC can grow and migrate in 3D hydrogel with the addition of FGF2, EGF, RGDSP and laminin. Further developments are necessary to form a homogenous tissue containing retinal cells.

TACI, unlike BAFF-R, is solely activated by oligomeric BAFF and APRIL to support survival of activated B cells and plasmablasts.

The cytokine BAFF binds to the receptors TACI, BCMA, and BAFF-R on B cells, whereas APRIL binds to TACI and BCMA only. The signaling properties of soluble trimeric BAFF (BAFF 3-mer) were compared with those of higher-order BAFF oligomers. All forms of BAFF bound BAFF-R and TACI, and elicited BAFF-R-dependent signals in primary B cells. In contrast, signaling through TACI in mature B cells or plasmablasts was only achieved by higher-order BAFF and APRIL oligomers, all of which were also po-tent activators of a multimerization-dependent reporter signaling pathway. These results indicate that, although BAFF-R and TACI can provide B cells with similar signals, only BAFF-R, but not TACI, can respond to soluble BAFF 3-mer, which is the main form of BAFF found in circulation. BAFF 60-mer, an efficient TACI agonist, was also detected in plasma of BAFF transgenic and nontransgenic mice and was more than 100-fold more active than BAFF 3-mer for the activation of multimerization-dependent signals. TACI supported survival of activated B cells and plasmablasts in vitro, providing a rational basis to explain the immunoglobulin deficiency reported in TACI-deficient persons.

Research resource: the dynamic transcriptional profile of sertoli cells during the progression of spermatogenesis.

Sertoli cells (SCs), the only somatic cells within seminiferous tubules, associate intimately with developing germ cells. They not only provide physical and nutritional support but also secrete factors essential to the complex developmental processes of germ cell proliferation and differentiation. The SC transcriptome must therefore adapt rapidly during the different stages of spermatogenesis. We report comprehensive genome-wide expression profiles of pure populations of SCs isolated at 5 distinct stages of the first wave of mouse spermatogenesis, using RNA sequencing technology. We were able to reconstruct about 13 901 high-confidence, nonredundant coding and noncoding transcripts, characterized by complex alternative splicing patterns with more than 45% comprising novel isoforms of known genes. Interestingly, roughly one-fifth (2939) of these genes exhibited a dynamic expression profile reflecting the evolving role of SCs during the progression of spermatogenesis, with stage-specific expression of genes involved in biological processes such as cell cycle regulation, metabolism and energy production, retinoic acid synthesis, and blood-testis barrier biogenesis. Finally, regulatory network analysis identified the transcription factors endothelial PAS domain-containing protein 1 (EPAS1/Hif2α), aryl hydrocarbon receptor nuclear translocator (ARNT/Hif1β), and signal transducer and activator of transcription 1 (STAT1) as potential master regulators driving the SC transcriptional program. Our results highlight the plastic transcriptional landscape of SCs during the progression of spermatogenesis and provide valuable resources to better understand SC function and spermatogenesis and its related disorders, such as male infertility.

Transfer of ultrasmall iron oxide nanoparticles from human brain-derived endothelial cells to human glioblastoma cells.

Nanoparticles (NPs) are being used or explored for the development of biomedical applications in diagnosis and therapy, including imaging and drug delivery. Therefore, reliable tools are needed to study the behavior of NPs in biological environment, in particular the transport of NPs across biological barriers, including the blood-brain tumor barrier (BBTB), a challenging question. Previous studies have addressed the translocation of NPs of various compositions across cell layers, mostly using only one type of cells. Using a coculture model of the human BBTB, consisting in human cerebral endothelial cells preloaded with ultrasmall superparamagnetic iron oxide nanoparticles (USPIO NPs) and unloaded human glioblastoma cells grown on each side of newly developed ultrathin permeable silicon nitride supports as a model of the human BBTB, we demonstrate for the first time the transfer of USPIO NPs from human brain-derived endothelial cells to glioblastoma cells. The reduced thickness of the permeable mechanical support compares better than commercially available polymeric supports to the thickness of the basement membrane of the cerebral vascular system. These results are the first report supporting the possibility that USPIO NPs could be directly transferred from endothelial cells to glioblastoma cells across a BBTB. Thus, the use of such ultrathin porous supports provides a new in vitro approach to study the delivery of nanotherapeutics to brain cancers. Our results also suggest a novel possibility for nanoparticles to deliver therapeutics to the brain using endothelial to neural cells transfer.

Directed integration of new insulated lentiviral vectors to heterochromatin towards safer gene transfer to stem cells

The need for better gene transfer systems towards improved risk=benefit balance for patients remains a major challenge in the clinical translation of gene therapy (GT). We have investigated the improvement of integrating vectors safety in combining (i) new short synthetic genetic insulator elements (GIE) and (ii) directing genetic integration to heterochromatin. We have designed SIN-insulated retrovectors with two candidate GIEs and could identify a specific combination of insulator 2 repeats which translates into best functional activity, high titers and boundary effect in both gammaretro (p20) and lentivectors (DCaro4) (see Duros et al, abstract ibid). Since GIEs are believed to shield the transgenic cassette from inhibitory effects and silencing, DCaro4 has been further tested with chimeric HIV-1 derived integrases which comprise C-ter chromodomains targeting heterochromatin through either histone H3 (ML6chimera) or methylatedCpGislands (ML10). With DCaro4 only and both chimeras, a homogeneous expression is evidenced in over 20% of the cells which is sustained over time. With control lentivectors, less than 2% of cells express GFP as compared to background using a control double-mutant in both catalytic and ledgf binding-sites; in addition, a two-times increase of expression can be induced with histone deacetylase inhibitors. Our approach could significantly reduce integration into open chromatin sensitive sites in stem cells at the time of transduction, a feature which might significantly decrease subsequent genotoxicity, according to X-SCIDs patients data.Work performed with the support of EC-DG research within the FP6-Network of Excellence, CLINIGENE: LSHB-CT-2006-018933

Persistent inhibition of FLIP(L) expression by lentiviral small hairpin RNA delivery restores death-receptor-induced apoptosis in neuroblastoma cells.

Neuroblastoma represents the most common and deadly solid tumour of childhood, which disparate biological and clinical behaviour can be explained by differential regulation of apoptosis. To understand mechanisms underlying death resistance in neuroblastoma cells, we developed small hairpin of RNA produced by lentiviral vectors as tools to selectively interfere with FLIP(L), a major negative regulator of death receptor-induced apoptosis. Such tools revealed highly efficient in interfering with FLIP(L) expression and function as they almost completely repressed endogenous and/or exogenously overexpressed FLIP(L) protein and fully reversed FLIP(L)-mediated TRAIL resistance. Moreover, interference with endogenous FLIP(L) and FLIP(S) significantly restored FasL sensitivity in SH-EP neuroblastoma cell line. These results reveal the ability of lentivirus-mediated shRNAs to specifically and persistently interfere with FLIP expression and support involvement of FLIP in the regulation of death receptor-mediated apoptosis in neuroblastoma cells. Combining such tools with other therapeutic modalities may improve treatment of resistant tumours such as neuroblastoma.

Intracortical connectivity of layer VI pyramidal cells in the barrel cortex of mice

ABSTRACT : The whisker-to-barrel pathway of rodents is formed by a series of somatotopic projections from the mystacial whisker follicles to the layer IV of the primary somatosensory cortex such that each follicle corresponds to a cluster of cortical neurons called barrel. Barrels are present in layer IV but form part of functional columns that comprise the entire depth of the somatosensory cortex. Interestingly, the cortex of the barrelless mouse strain (BRL) is organized such a manner that thalamocortical afferents do not remodel their projections in layer IV and barrels fail to appear. Nevertheless, functionally, a columnar organization persists, indicating that functional columns are not only provided by thalamocortical projections and layer IV cells. Since in the visual cortex of cats, layer VI cells contribute to the response properties of layer IV neurons, we wonder whether layer VI pyramidal cells could contribute to the columnar organization of the primary somatosensory cortex of mice. To address -this question, we morphologically analyzed the distribution of intracortical axon collaterals of layer VI neurons after in-vivo juxtacellular injections of biocytin in the C2 barrel column. Injected hemispheres were tangentially serial cut and intracortical collaterals of individual layer VI neurons were reconstructed at the light microscopic level. The position of axonal boutons was recorded to evaluate the distribution of presumed synaptic contacts. In normal (NOR) mice, cluster analysis shows that layer VI pyramidal cells can be classified in four statistically different clusters of neurons. Moreover, we assume that two classes are formed by cortico-cortical neurons and two classes are formed by cortico-thalamic neurons. Looking at the direction of the main axon in the white matter, we noticed that its orientation correlates perfectly with the type of neuron: cortico-cortical neurons send main axon medially whereas cortico-thalamic neurons send main axon laterally. Performing the same study in the BRL strain, we showed that the BRL mutation affects layer VI pyramidal cells tangentially and radially: the effects of the mutation are illustrated by a significant decrease of the index of colurnnarization and a significant decrease of percentage of boutons in granular and supragranular layers comparing to NOR neurons. In spite of these differences, the same four classes of layer VI neurons have been found in BRL mice. Using a tangential analysis of the boutons distribution, we showed that putative synapses are distributed mainly in the C2 barrel column. This was observed for each layer, type of neuron, cluster or strain, indicating that layer VI pyramidal cells could participate to the functional columnar organization of the barrel cortex. To determine post-synaptic partners of layer VI neurons in layer IV, we conducted an ultrastructural analysis of layer VI-to-IV contacts. We showed that synapses principally occur on spines and spiny dendritic shafts, supposed to belong to excitatory neurons. We furthermore showed that pre-synaptic elements are significantly different between en passant and terminaux contacts, which support hypothesis that terminaux boutons should show longer duration of facilitation than en passant boutons. RÉSUMÉ : Le «whisker-to-barrel pathway» des rongeurs est caractérisé par une série de projections somatotopiques depuis les follicules des moustaches ('whiskers') jusqu'à la couche IV de l'aire somatosensorielle primaire, de telle façon que chaque follicule corresponde à un groupe de neurones corticaux appelés tonneaux (`barrels'). Les tonneaux sont seulement présents en couche IV mais font partie de colonnes fonctionnelles qui s'étendent sur toute la profondeur du cortex somatosensoriel. Chez les souris mutantes barrelless (BRL), le cortex somatosensoriel est organisé de façon telle que lés afférences thalamocorticales ne remodellent pas leurs projections en couche IV et que les tonneaux n'apparaissent pas. Fonctionnellement, pourtant, une organisation en colonnes persiste, ce qui indique que les colonnes fonctionnelles ne sont pas uniquement produites par les projections thalamocorticales et par les cellules de la couche IV. Puisque les cellules de la couche VI contribuent à influencer les réponses des cellules de la couche IV dans le cortex visuel du chat, nous nous sommes demandé si ces cellules ne pourraient pas aussi contribuer à l'organisation en colonnes du cortex somatosensoriel primaire de la souris. Pour répondre à cette question, nous avons analysé de façon morphologique la distribution intracorticale des collatéraux axonaux de neurones de la couche VI. Suite à des injections juxtacellulaires de biocytine in-vivo dans la colonne C2, les hémisphères cérébraux ont été tangentiellement coupés en série et les collatéraux intracorticaux des neurones de la couche VI ont été reconstruits en microscopie optique. La position des boutons axonaux a aussi été enregistrée pour évaluer la distribution des contacts synpptiques potentiels. Chez les souris NOR, une analyse multivariée montre que les cellules pyramidales de la couche VI sont distribuées en quatre classes. Deux de ces classes sont probablement formées de neurons cortico-corticaux, alors que les deux autres sont probablement formées de neurones corticothalamiques. En observant la direction de l'axone principal dans la matière blanche, nous avons noté que son orientation est parfaitement corrélée avec le type supposé de neurone : les neurones corticocorticaux envoient leurs axones principaux médiallement, alors que les neurons cortico-thalamiques envoient leurs axones principaux latéralement. En menant la même étude chez les souris BRL, nous avons montré que la mutation affecte les cellules pyramidales de la couche VI de façon tangentielle, mais aussi radiaire : les effets de 1a mutation se traduisent par une diminution significative de l'index de « columnarization » et de la connectivité en couches granulaire et supragranulaire. Malgré ces différences, les quatre mêmes classes de neurones ont été retrouvées. En utilisant une analyse tangentielle de la distribution des boutons, nous avons montré que les synapses potentielles sont distribuées principalement dans la colonne C2. Cette observation a été faite dans chaque couche, chaque type de neurones, chaque classe de neurones et chaque souche de souris, indicant que les cellules de la couche VI participent certainement à l'organisation en colonne du cortex somatosensoriel. Pour déterminer les partenaires post-synaptiques des cellules de la couche VI en couche IV, nous avons conduit une analyse ultrastructurelle de ces contacts. Nous avons montré que les synapses interviennent principalement sur les épines et sur les dendrites supposés appartenir à des cellules excitatrices. Nous avons aussi montré que les éléments pré-synaptiques de ces synapses sont significativement differents selon le type de bouton, en passant ou terminal, ce qui supporte l'hypothèse que les boutons terminaux seraient capables d'une plus longue facilitation.

Molecular pathways involved in the antineoplastic effects of calcitriol on insulinoma cells.

We have previously reported that in tumorigenic pancreatic beta-cells, calcitriol exerts a potent antitumorigenic effect by inducing apoptosis, cell growth inhibition, and reduction of solid beta-cell tumors. Here we have studied the molecular pathways involved in the antineoplastic activity of calcitriol on mouse insulinoma beta TC(3) cells, mouse insulinoma beta TC expressing or not expressing the oncogene p53, and beta TC-tet cells overexpressing or not the antiapoptotic gene Bcl2. Our results indicate that calcitriol-induced apoptosis was dependent on the function of p53 and was associated with a biphasic increase in protein levels of transcription factor nuclear factor-kappa B. Calcitriol decreased cell viability by about 40% in p53-retaining beta TC and in beta TC(3) cells; in contrast, beta TC p53(-/-) cells were only minimally affected. Calcitriol-induced cell death was regulated by members of the Bcl-2 family of apoptosis regulatory proteins, as shown by calcitriol-induced up-regulation of proapoptotic Bax and Bak and the lack of calcitriol-induced cytotoxicity in Bcl-2-overexpressing insulinoma cells. Moreover, calcitriol-mediated arrest of beta TC(3) cells in the G(1) phase of the cell cycle was associated with the abnormal expression of p21 and G(2)/M-specific cyclin B2 genes and involved the DNA damage-inducible factor GADD45. Finally, in beta TC(3) cells, calcitriol modulated the expression of IGF-I and IGF-II genes. In conclusion, these findings contribute to the understanding of the antitumorigenic effects of calcitriol on tumorigenic pancreatic beta-cells and further support the rationale of its utilization in the treatment of patients with malignant insulinomas.

Education of Murine NK Cells Requires Both cis and trans Recognition of MHC Class I Molecules.

Although NK cells use invariant receptors to identify diseased cells, they nevertheless adapt to their environment, including the presence of certain MHC class I (MHC-I) molecules. This NK cell education, which is mediated by inhibitory receptors specific for MHC-I molecules, changes the responsiveness of activating NK cell receptors (licensing) and modifies the repertoire of MHC-I receptors used by NK cells. The fact that certain MHC-I receptors have the unusual capacity to recognize MHC-I molecules expressed by other cells (trans) and by the NK cell itself (cis) has raised the question regarding possible contributions of the two types of interactions to NK cell education. Although the analysis of an MHC-I receptor variant suggested a role for cis interaction for NK cell licensing, adoptive NK cell transfer experiments supported a key role for trans recognition. To reconcile some of these findings, we have analyzed the impact of cell type-specific deletion of an MHC-I molecule and of a novel MHC-I receptor variant on the education of murine NK cells when these mature under steady-state conditions in vivo. We find that MHC-I expression by NK cells (cis) and by T cells (trans), and MHC-I recognition in cis and in trans, are both needed for NK cell licensing. Unexpectedly, modifications of the MHC-I receptor repertoire are chiefly dependent on cis binding, which provides additional support for an essential role for this unconventional type of interaction for NK cell education. These data suggest that two separate functions of MHC-I receptors are needed to adapt NK cells to self-MHC-I.

Evaluation of somatic cell variants deficient in glycosylphosphatidyl-inositol anchoring as candidates for genetic correction.

Many cell surface glycoproteins are anchored in the lipid bilayer by a glycosylphosphatidyl-inositol (GPI) structure. Recently, a number of cell lines which are deficient in the biosynthesis and/or addition of this anchor have been described. In this report, we summarize the current knowledge on these lines and discuss their potential use to isolate the genes involved in the GPI anchor biosynthetic pathway with a specific emphasis on L cell fibroblasts.

Autophagy promotes survival of retinal ganglion cells after optic nerve axotomy in mice.

Autophagy is an essential recycling pathway implicated in neurodegeneration either as a pro-survival or a pro-death mechanism. Its role after axonal injury is still uncertain. Axotomy of the optic nerve is a classical model of neurodegeneration. It induces retinal ganglion cell death, a process also occurring in glaucoma and other optic neuropathies. We analyzed autophagy induction and cell survival following optic nerve transection (ONT) in mice. Our results demonstrate activation of autophagy shortly after axotomy with autophagosome formation, upregulation of the autophagy regulator Atg5 and apoptotic death of 50% of the retinal ganglion cells (RGCs) after 5 days. Genetic downregulation of autophagy using knockout mice for Atg4B (another regulator of autophagy) or with specific deletion of Atg5 in retinal ganglion cells, using the Atg5(flox/flox) mice reduces cell survival after ONT, whereas pharmacological induction of autophagy in vivo increases the number of surviving cells. In conclusion, our data support that autophagy has a cytoprotective role in RGCs after traumatic injury and may provide a new therapeutic strategy to ameliorate retinal diseases.

Neuropeptide-inducible upregulation of proteasome activity precedes nuclear factor kappa B activation in androgen-independent prostate cancer cells.

ABSTRACT: BACKGROUND: Upregulation of nuclear factor kappa B (NFκB) activity and neuroendocrine differentiation are two mechanisms known to be involved in prostate cancer (PC) progression to castration resistance. We have observed that major components of these pathways, including NFκB, proteasome, neutral endopeptidase (NEP) and endothelin 1 (ET-1), exhibit an inverse and mirror image pattern in androgen-dependent (AD) and -independent (AI) states in vitro. METHODS: We have now investigated for evidence of a direct mechanistic connection between these pathways with the use of immunocytochemistry (ICC), western blot analysis, electrophoretic mobility shift assay (EMSA) and proteasome activity assessment. RESULTS: Neuropeptide (NP) stimulation induced nuclear translocation of NFκB in a dose-dependent manner in AI cells, also evident as reduced total inhibitor κB (IκB) levels and increased DNA binding in EMSA. These effects were preceded by increased 20 S proteasome activity at lower doses and at earlier times and were at least partially reversed under conditions of NP deprivation induced by specific NP receptor inhibitors, as well as NFκB, IκB kinase (IKK) and proteasome inhibitors. AD cells showed no appreciable nuclear translocation upon NP stimulation, with less intense DNA binding signal on EMSA. CONCLUSIONS: Our results support evidence for a direct mechanistic connection between the NPs and NFκB/proteasome signaling pathways, with a distinct NP-induced profile in the more aggressive AI cancer state.