RESUMEN
Bone marrow hematopoietic stem cells (HSCs) are crucial to maintain lifelong production of all blood cells. Although HSCs divide infrequently, it is thought that the entire HSC pool turns over every few weeks, suggesting that HSCs regularly enter and exit cell cycle. Here, we combine flow cytometry with label-retaining assays (BrdU and histone H2B-GFP) to identify a population of dormant mouse HSCs (d-HSCs) within the lin(-)Sca1+cKit+CD150+CD48(-)CD34(-) population. Computational modeling suggests that d-HSCs divide about every 145 days, or five times per lifetime. d-HSCs harbor the vast majority of multilineage long-term self-renewal activity. While they form a silent reservoir of the most potent HSCs during homeostasis, they are efficiently activated to self-renew in response to bone marrow injury or G-CSF stimulation. After re-establishment of homeostasis, activated HSCs return to dormancy, suggesting that HSCs are not stochastically entering the cell cycle but reversibly switch from dormancy to self-renewal under conditions of hematopoietic stress.
Asunto(s)
Células Madre Adultas/citología , Células Madre Hematopoyéticas/citología , Células Madre Adultas/fisiología , Animales , Antígenos de Diferenciación/metabolismo , Médula Ósea/fisiología , Bromouracilo/análogos & derivados , Fluorouracilo/metabolismo , Proteínas Fluorescentes Verdes , Células Madre Hematopoyéticas/fisiología , Homeostasis , Ratones , Ratones Transgénicos , Uridina/análogos & derivados , Uridina/metabolismoRESUMEN
The protease activity of the paracaspase Malt1 has recently gained interest as a drug target for immunomodulation and the treatment of diffuse large B-cell lymphomas. To address the consequences of Malt1 protease inactivation on the immune response in vivo, we generated knock-in mice expressing a catalytically inactive C472A mutant of Malt1 that conserves its scaffold function. Like Malt1-deficient mice, knock-in mice had strong defects in the activation of lymphocytes, NK and dendritic cells, and the development of B1 and marginal zone B cells and were completely protected against the induction of autoimmune encephalomyelitis. Malt1 inactivation also protected the mice from experimental induction of colitis. However, Malt1 knock-in mice but not Malt1-deficient mice spontaneously developed signs of autoimmune gastritis that correlated with an absence of Treg cells, an accumulation of T cells with an activated phenotype and high serum levels of IgE and IgG1. Thus, removal of the enzymatic activity of Malt1 efficiently dampens the immune response, but favors autoimmunity through impaired Treg development, which could be relevant for therapeutic Malt1-targeting strategies.
Asunto(s)
Autoinmunidad/genética , Caspasas/metabolismo , Colitis/inmunología , Proteínas de Unión al ADN/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Proteínas de Neoplasias/metabolismo , Traslado Adoptivo , Análisis de Varianza , Animales , Caspasas/genética , Colitis/patología , Cartilla de ADN/genética , Proteínas de Unión al ADN/genética , Encefalomielitis Autoinmune Experimental/patología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Técnicas de Sustitución del Gen , Silenciador del Gen , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Inmunohistoquímica , Células Asesinas Naturales/inmunología , Ratones , Ratones Noqueados , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Proteínas de Neoplasias/genética , Mutación Puntual/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The paracaspase MALT1 has a central role in the activation of lymphocytes and other immune cells including myeloid cells, mast cells and NK cells. MALT1 activity is required not only for the immune response, but also for the development of natural Treg cells that keep the immune response in check. Exaggerated MALT1 activity has been associated with the development of lymphoid malignancies, and recently developed MALT1 inhibitors show promising anti-tumor effects in xenograft models of diffuse large B cell lymphoma. In this review, we provide an overview of the present understanding of MALT1's function, and discuss possibilities for its therapeutic targeting based on recently developed inhibitors and animal models.
Asunto(s)
Caspasas/fisiología , Proteínas de Neoplasias/fisiología , Secuencias de Aminoácidos , Animales , Linfocitos B/metabolismo , Linfocitos B/fisiología , Caspasas/metabolismo , Humanos , Inmunomodulación/genética , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/fisiología , Linfoma de Células B Grandes Difuso/genética , Modelos Moleculares , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , FN-kappa B/metabolismo , FN-kappa B/fisiología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción AP-1/fisiologíaRESUMEN
Cell-intrinsic checkpoints limit the proliferative capacity of primary cells in response to telomere dysfunction. It is not known, however, whether telomere dysfunction contributes to cell-extrinsic alterations that impair stem cell function and organ homeostasis. Here we show that telomere dysfunction provokes defects of the hematopoietic environment that impair B lymphopoiesis but increase myeloid proliferation in aging telomerase knockout (Terc(-/-)) mice. Moreover, the dysfunctional environment limited the engraftment of transplanted wild-type hematopoietic stem cells (HSCs). Dysfunction of the hematopoietic environment was age dependent and correlated with progressive telomere shortening in bone marrow stromal cells. Telomere dysfunction impaired mesenchymal progenitor cell function, reduced the capacity of bone marrow stromal cells to maintain functional HSCs, and increased the expression of various cytokines, including granulocyte colony-stimulating factor (G-CSF), in the plasma of aging mice. Administration of G-CSF to wild-type mice mimicked some of the defects seen in aging Terc(-/-) mice, including impairment of B lymphopoiesis and HSC engraftment. Conversely, inhibition of G-CSF improved HSC engraftment in aged Terc(-/-) mice. Taken together, these results show that telomere dysfunction induces alterations of the environment that can have implications for organismal aging and cell transplantation therapies.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/patología , Homeostasis/genética , Telómero/fisiología , Animales , Apoptosis/genética , Linfocitos B/patología , Proliferación Celular , Células Cultivadas , Senescencia Celular/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/patología , ARN/genética , Telomerasa/deficiencia , Telomerasa/genética , Telómero/genética , Regulación hacia Arriba/genéticaRESUMEN
The protease activity of the paracaspase Malt1 contributes to antigen receptor-mediated lymphocyte activation and lymphomagenesis. Malt1 activity is required for optimal NF-κB activation, but little is known about the responsible substrate(s). Here we report that Malt1 cleaved the NF-κB family member RelB after Arg-85. RelB cleavage induced its proteasomal degradation and specifically controlled DNA binding of RelA- or c-Rel-containing NF-κB complexes. Overexpression of RelB inhibited expression of canonical NF-κB target genes and led to impaired survival of diffuse large B-cell lymphoma cell lines characterized by constitutive Malt1 activity. These findings identify a central role for Malt1-dependent RelB cleavage in canonical NF-κB activation and thereby provide a rationale for the targeting of Malt1 in immunomodulation and cancer treatment.
Asunto(s)
Caspasas/fisiología , Linfocitos/metabolismo , Linfoma de Células B Grandes Difuso/metabolismo , FN-kappa B/metabolismo , Proteínas de Neoplasias/fisiología , Factor de Transcripción ReIB/metabolismo , Línea Celular Tumoral , Humanos , Activación de Linfocitos , Linfoma de Células B Grandes Difuso/etiología , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Factor de Transcripción ReIA/metabolismoRESUMEN
T cell receptor (TCR)-based immunotherapy has emerged as a promising therapeutic approach for the treatment of patients with solid cancers. Identifying peptide-human leukocyte antigen (pHLA) complexes highly presented on tumors and rarely expressed on healthy tissue in combination with high-affinity TCRs that when introduced into T cells can redirect T cells to eliminate tumor but not healthy tissue is a key requirement for safe and efficacious TCR-based therapies. To discover promising shared tumor antigens that could be targeted via TCR-based adoptive T cell therapy, we employed population-scale immunopeptidomics using quantitative mass spectrometry across ~1500 tumor and normal tissue samples. We identified an HLA-A*02:01-restricted pan-cancer epitope within the collagen type VI α-3 (COL6A3) gene that is highly presented on tumor stroma across multiple solid cancers due to a tumor-specific alternative splicing event that rarely occurs outside the tumor microenvironment. T cells expressing natural COL6A3-specific TCRs demonstrated only modest activity against cells presenting high copy numbers of COL6A3 pHLAs. One of these TCRs was affinity-enhanced, enabling transduced T cells to specifically eliminate tumors in vivo that expressed similar copy numbers of pHLAs as primary tumor specimens. The enhanced TCR variants exhibited a favorable safety profile with no detectable off-target reactivity, paving the way to initiate clinical trials using COL6A3-specific TCRs to target an array of solid tumors.
Asunto(s)
Inmunoterapia Adoptiva , Receptores de Antígenos de Linfocitos T , Linfocitos T , Antígenos de Neoplasias , Línea Celular Tumoral , Tratamiento Basado en Trasplante de Células y Tejidos , Humanos , Inmunoterapia Adoptiva/métodos , Proteómica , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/uso terapéuticoRESUMEN
In the mouse, over the last 20 years, a set of cell-surface markers and activities have been identified, enabling the isolation of bone marrow (BM) populations highly enriched in hematopoietic stem cells (HSCs). These HSCs have the ability to generate multiple lineages and are capable of long-term self-renewal activity such that they are able to reconstitute and maintain a functional hematopoietic system after transplantation into lethally irradiated recipients. Using single-cell reconstitution assays, various marker combinations can be used to achieve a functional HSC purity of almost 50%. Here we have used the differential expression of six of these markers (Sca1, c-Kit, CD135, CD48, CD150, and CD34) on lineage-depleted BM to refine cell hierarchies within the HSC population. At the top of the hierarchy, we propose a dormant HSC population (Lin(-)Sca1(+)c-Kit(+) CD48(-)CD150(+)CD34(-)) that gives rise to an active self-renewing CD34(+) HSC population. HSC dormancy, as well as the balance between self-renewal and differentiation activity, is at least, in part, controlled by the stem cell niches individual HSCs are attached to. Here we review the current knowledge about HSC niches and propose that dormant HSCs are located in niches at the endosteum, whereas activated HSCs are in close contact to sinusoids of the BM microvasculature.
Asunto(s)
Células Madre Hematopoyéticas/citología , Animales , Médula Ósea/metabolismo , Células de la Médula Ósea/citología , Diferenciación Celular , Regulación de la Expresión Génica , Ratones , Modelos Biológicos , Modelos Genéticos , Osteoblastos/metabolismo , FenotipoRESUMEN
The mitogen-activated protein kinase signalling pathway is a central regulator of tumor growth, which is constitutively activated in chemically induced mouse liver tumors. In about 30-50% of cases this effect can be related to activation of the Ha-ras gene by point mutations, whereas in the remaining cases mutations may occur in other members within this pathway, such as Raf kinases. Recently, B-raf has been shown to be frequently mutated in human melanomas and certain other cancers, with a V599E amino-acid change representing the most predominant mutation type. We now screened 82 N-nitrosodiethylamine-induced liver tumors from C3H/He mice for mutations within the hotspot positions in the Ha-ras and B-raf genes. About 50% (39/82) of tumors showed Ha-ras codon 61 mutations and 16 tumors ( approximately 20%) harbored mutations at codon 624 of the B-raf gene, which corresponds to codon 599 in human B-raf. None of the tumors was mutated in both Ha-ras and B-raf. The high prevalence of Ha-ras and B-raf mutations in mouse liver tumors is in striking contrast to human hepatocellular cancers which very infrequently harbor mutations in the two genes. These fundamental differences between the biology of liver tumors in mice and man may be of toxicological relevance.
Asunto(s)
Genes ras/genética , Neoplasias Hepáticas Experimentales/genética , Ratones/genética , Mutación Puntual/genética , Proteínas Proto-Oncogénicas B-raf/genética , Animales , Transformación Celular Neoplásica/genética , ADN de Neoplasias/genética , Dietilnitrosamina , Humanos , Neoplasias Hepáticas Experimentales/inducido químicamente , Ratones Endogámicos C3H , Análisis de Secuencia de ADNRESUMEN
In a previous microarray expression analysis, Rex3, a gene formerly not linked to tumor formation, was found to be highly overexpressed in both Ctnnb1-(beta-Catenin) and Ha-ras-mutated mouse liver tumors. Subsequent analyses by in situ hybridization and real-time PCR confirmed a general liver tumor-specific overexpression of the gene (up to 400-fold). To investigate the role of Rex3 in liver tumors, hepatoma cells were transfected with FLAG- and Myc-tagged Rex3 expression vectors. Rex3 was shown to be exclusively localized to the cytoplasm, as determined by fluorescence microscopy and Western blotting. However, forced overexpression of Rex3 did not significantly affect proliferation or stress-induced apoptosis of transfected mouse hepatoma cells. Rex3 mRNA was determined in primary hepatocytes in culture by real-time PCR. In primary mouse hepatocytes, expression of Rex3 increased while cells dedifferentiated in culture. This effect was abolished when hepatocytes were maintained in a differentiated state. Furthermore, expression of Rex3 decreased in mouse liver with age of mice and the expression profile was highly correlated to that of the tumor markers alpha-fetoprotein and H19. The findings suggest a role of Rex3 as a marker for hepatocyte differentiation/dedifferentiation processes and tumor formation.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Diferenciación Celular , Proteínas de Unión al ADN/biosíntesis , Hepatocitos/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Animales , Apoptosis , Western Blotting , Proliferación Celular , Electroforesis en Gel de Poliacrilamida , Hepatocitos/patología , Inmunohistoquímica , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones , Ratones Endogámicos C3H , ARN Mensajero/biosíntesis , Transfección , Células Tumorales CultivadasRESUMEN
The immune system and iron availability are intimately linked as appropriate iron supply is needed for cell proliferation, while excess iron, as observed in hemochromatosis, may reduce subsets of lymphocytes. We have tested the effects of a ferritin H gene deletion on lymphocytes. Mx-Cre mediated conditional deletion of ferritin H in bone marrow reduced the number of mature B cells and peripheral T cells in all lymphoid organs. FACS analysis showed an increase in the labile iron pool, enhanced reactive oxygen species formation and mitochondrial depolarization. The findings were confirmed by a B-cell specific deletion using Fth(lox/lox) ; CD19-Cre mice. Mature B cells were strongly under-represented in bone marrow and spleen of the deleted mice, whereas pre-B and immature B cells were not affected. Bone marrow B cells showed increased proliferation as judged by the number of cells in S and G2/M phase as well as BrdU incorporation. Upon in vitro culture with B-cell activating factor of the tumor necrosis factor family (BAFF), ferritin H-deleted spleen B cells showed lower survival rates than wild type cells. This was partially reversed with iron-chelator deferiprone. The loss of T cells was also confirmed by a T cell-specific deletion in Fth(lox/lox) ;CD4-Cre mice. Our data show that ferritin H is required for B and T cell survival by actively reducing the labile iron pool. They further suggest that natural B and T cell maturation is influenced by intracellular iron levels and possibly deregulated in iron excess or deprivation.
Asunto(s)
Apoferritinas/genética , Linfocitos B/citología , Proliferación Celular/genética , Hierro/metabolismo , Linfocitos T/citología , Animales , Linfocitos B/metabolismo , Recuento de Células , Diferenciación Celular/genética , Ratones , Ratones Noqueados , Especies Reactivas de Oxígeno/metabolismo , Bazo/citología , Bazo/metabolismo , Linfocitos T/metabolismoRESUMEN
In the bone marrow, specialized microenvironments, called niches, regulate hematopoietic stem cell (HSC) maintenance and function through a complex crosstalk between different cell types. Although in vivo studies have been instrumental to elucidate some of the mechanisms by which niches exert their function, the establishment of an in vitro model that recapitulates the fundamental interactions of the niche components in a controlled setting would be of great benefit. We have previously shown that freshly harvested bone marrow- or adipose tissue-derived cells can be cultured under perfusion within porous scaffolds, allowing the formation of an organized 3D stromal tissue, composed by mesenchymal and endothelial progenitors and able to support hematopoiesis. Here we describe 3D scaffold-based perfusion systems as potential models to reconstruct ex vivo the bone marrow stem cell niche. We discuss how several culture parameters, including scaffold properties, cellular makeup and molecular signals, can be varied and controlled to investigate the role of specific cues in affecting HSC fate. We then provide a perspective of how the system could be exploited to improve stem cell-based therapies and how the model can be extended toward the engineering of other specialized stromal niches.
Asunto(s)
Células de la Médula Ósea/citología , Técnicas de Cultivo de Célula/métodos , Nicho de Células Madre/citología , Andamios del Tejido , Animales , HumanosRESUMEN
The tumor suppressor p53 is frequently mutated in cancer. We have investigated the regulation of P53 in p53 wild type mouse hepatoma cells (line 55.1c), in p53 heterozygeously mutated cells (56.1b) and in p53 defective cells (lines 56.1d, 70.4 and HUH7) under various experimental settings. The basal levels of P53 were low in 55.1c cells, but nuclear accumulation occurred upon UV-irradiation. Similarly, UV-exposure induced stabilization of P53 in the heterozygeously p53 mutated 56.1b hepatoma cells. By contrast, the 3 hepatoma lines, which lack transcriptionally active P53, demonstrated high basal nuclear concentrations of P53 protein and, unexpectedly, showed loss of P53 upon UV-irradiation. Expression of p53 mRNA was also decreased in p53 defective cells after 24 hr post UV-irradiation, which may be linked to induction of apoptosis of the irradiated cells under these conditions. Other stressors like H2O2 also mediated a decrease in P53 concentration in p53 defective cells. This effect occurred at very low concentrations and was already detectable 1-2 hr after exposure of cells. There were no signs of apoptosis of H2O2-exposed cells at this time point and no significant changes in p53 mRNA or MDM2 level. These unexpected findings indicate a new aspect related to regulation of P53 stability in cells with a defect in the tumor suppressor protein.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/metabolismo , Mutación/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/fisiología , Apoptosis/efectos de la radiación , Western Blotting , Carcinoma Hepatocelular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Humanos , Inmunoprecipitación , Neoplasias Hepáticas/genética , Ratones , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/efectos de la radiación , Rayos UltravioletaRESUMEN
Chemically-induced mouse liver tumors harbor mutations in different oncogenes. About 50% of tumors contain activating mutations in the Ha-ras gene contain and about 20% of tumors show point mutations in the B-raf oncogene. We have investigated the gene expression profiles in tumors of the 2 genotypes by microarray analysis. In total, approximately 500 genes or expressed sequences were aberrantly expressed in tumors relative to normal liver tissue. Around two/thirds of them were significantly altered in both Ha-ras and B-raf mutated liver tumors, and most of the remaining genes showed at least qualitatively comparable changes in both tumor types. Several functional clusters were hypothesized in tumors of the 2 genotypes which involve alterations in a battery of genes encoding enzymes of lipid metabolism. The similarity in the patterns of global gene expression of Ha-ras and B-raf mutated liver tumors suggests that mutational activation of the 2 oncogenes results in activation of a common set of transcriptional regulators.
Asunto(s)
Genes ras/genética , Neoplasias Hepáticas Experimentales/genética , Proteínas Proto-Oncogénicas B-raf/genética , Alquilantes/toxicidad , Animales , Dietilnitrosamina/toxicidad , Expresión Génica , Perfilación de la Expresión Génica , Neoplasias Hepáticas Experimentales/inducido químicamente , Ratones , Ratones Endogámicos C3H , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Gene expression in hepatocytes within the liver lobule is differentially regulated along the portal to central axis; however, the mechanisms governing the processes of zonation within the lobule are unknown. A model for zonal heterogeneity in normal liver is proposed, based on observations of differential expression of genes in liver tumors from mice that harbor activating mutations in either Catnb (which codes for beta-catenin) or Ha-ras. According to the model, the regulatory control consists of two opposing signals, one delivered by endothelial cells of the central veins activating a beta-catenin-dependent pathway (retrograde signal), the other by blood-borne molecules activating Ras-dependent downstream cascades (anterograde signal). In conclusion, gradients of opposing signaling molecules along the portocentral axis determine the pattern of enzymes and other proteins expressed in hepatocytes of the periportal and pericentral domains of the liver lobule.
Asunto(s)
Expresión Génica/genética , Genes ras/genética , Neoplasias Hepáticas/genética , Hígado/metabolismo , beta Catenina/genética , Animales , Cadherinas/biosíntesis , Cadherinas/genética , Modelos Animales de Enfermedad , Glutamato-Amoníaco Ligasa/genética , Hepatocitos/metabolismo , Hepatocitos/patología , Hígado/patología , Neoplasias Hepáticas/patología , Ratones , Modelos Animales , Transducción de Señal , beta Catenina/biosíntesisRESUMEN
Mouse models are important tools in toxicologic research. Differences between species in pathways contributing to tumor development, however, raise the question in how far mouse models are valid for human risk assessment. One striking difference relates to the frequency of spontaneous liver cancer which is high in certain mouse strains but rather low in humans. Similarly, mutation frequencies in cancer genes are characteristically different, i.e. P53 mutations are frequent in human but very rare in murine liver tumors, whereas Ras genes are often mutated in mouse liver tumors but hardly ever in human liver cancers. Since P53 has been shown to control oncogenic RAS in human cells, we hypothesized that this function of the tumor suppressor could differ in mouse hepatocytes. To test this hypothesis, we used hupki (human p53 knock-in) mice which carry a partly humanized P53 sequence (P53KI). In this study, we report the results of the first hepatocarcinogenesis experiment with this strain of mice. Mice of the genotypes P53KI/KI, P53WT/KI and P53WT/WT were treated with N-nitrosodiethylamine at 2 weeks of age and killed 35 weeks later. The frequency of liver tumors and glucose-6-phosphatase-altered liver lesions was almost identical in all three P53 genotypes and approximately 40-50% of liver tumors showed activating mutations in codon 61 of the Ha-Ras gene independent of genotype. Moreover, only very few P53-positive lesions were observed but without nuclear localization of the protein, suggesting the absence of P53 mutations. These data suggest that the hupki allele behaves like its murine ortholog in mouse hepatocarcinogenesis.