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1.
Blood ; 121(10): 1814-8, 2013 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-23319569

RESUMEN

Bone marrow (BM) provides chemoprotection for acute lymphoblastic leukemia (ALL) cells, contributing to lack of efficacy of current therapies. Integrin alpha4 (alpha4) mediates stromal adhesion of normal and malignant B-cell precursors, and according to gene expression analyses from 207 children with minimal residual disease, is highly associated with poorest outcome. We tested whether interference with alpha4-mediated stromal adhesion might be a new ALL treatment. Two models of leukemia were used, one genetic (conditional alpha4 ablation of BCR-ABL1 [p210(+)] leukemia) and one pharmacological (anti-functional alpha4 antibody treatment of primary ALL). Conditional deletion of alpha4 sensitized leukemia cell to nilotinib. Adhesion of primary pre-B ALL cells was alpha4-dependent; alpha4 blockade sensitized primary ALL cells toward chemotherapy. Chemotherapy combined with Natalizumab prolonged survival of NOD/SCID recipients of primary ALL, suggesting adjuvant alpha4 inhibition as a novel strategy for pre-B ALL.


Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Resistencia a Antineoplásicos , Proteínas de Fusión bcr-abl/fisiología , Integrina alfa4/química , Neoplasia Residual/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Médula Ósea/patología , Adhesión Celular , Niño , Citometría de Flujo , Humanos , Integrasas/metabolismo , Integrina alfa4/genética , Integrina alfa4/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Natalizumab , Neoplasia Residual/metabolismo , Neoplasia Residual/mortalidad , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidad , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Células del Estroma/patología
2.
Toxicology ; 259(3): 91-6, 2009 May 17.
Artículo en Inglés | MEDLINE | ID: mdl-19428948

RESUMEN

The aryl hydrocarbon receptor (AhR) mediates toxicity of a variety of environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) and dioxins. However, the underlying mechanisms and genetic programmes regulated by AhR to cause adverse effects but also to counteract poisoning are still poorly understood. Here we analysed the effects of two AhR ligands, benzo[a]pyrene (B[a]P), a DNA damaging tumour initiator and promotor and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a pure tumour promoter, on cell survival and on nucleotide excision repair (NER) gene expression. NER deals with so called "bulky" DNA adducts including those generated by enzymatically activated B[a]P. Therefore, the hypothesis that AhR may enhance NER gene expression to trigger DNA repair in the presence of genotoxic AhR ligands was tested. Furthermore, we investigated a potential cytoprotective effect of AhR activation by the non-genotoxic ligand TCDD against cell death induced by various genotoxins. Finally, the actions of genotoxins themselves on NER gene expression were studied. As a cell culture model we used mouse hepatoma cells (Hepa-c7) proficient for AhR and its partner protein ARNT as well as subclones deficient in AhR (Hepa-c12) or ARNT (Hepa-c4) to study involvement of AhR and ARNT in response to B[a]P and TCDD. Indeed, the mRNA levels of the two NER genes XP-C and DNA polymerase kappa were increased by B[a]P and TCDD, however, this was not accompanied by an increase in the amount of the respective proteins. Pretreatment of cells with TCDD did not reduce cytotoxicity induced by various genotoxins. Thus, in Hepa-c7 cells AhR has no major effects on the expression of these crucial NER proteins and does not prevent genotoxin-provoked cell death. As expected, the genotoxins B[a]P and cis-platin led to p53 accumulation and induction of its target p21. Interestingly, however, NER gene expression was not enhanced but rather decreased. As two NER genes, XP-C and DNA damage binding protein ddb2, are up-regulated by p53 and ultraviolet radiation in human cells these findings suggest cell type, species or lesion specific actions of p53 on DNA repair gene expression. Importantly, in cells with damaged DNA up-regulation of p53 may not suffice to enhance DNA repair gene expression.


Asunto(s)
Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Reparación del ADN/fisiología , Mutágenos/toxicidad , Receptores de Hidrocarburo de Aril/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Reparación del ADN/genética , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Contaminantes Ambientales/metabolismo , Contaminantes Ambientales/toxicidad , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Genes p53 , Neoplasias Hepáticas Experimentales , Ratones , Mutágenos/metabolismo
3.
Exp Hematol ; 78: 35-45, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31562901

RESUMEN

Endocannabinoids are lipid mediators that signal via several seven-transmembrane domain G protein-coupled receptors. The endocannabinoid receptor CB2 is expressed on blood cells, including stem cells, and mediates the effects of cannabinoids on the immune system. The role of the endocannabinoid system in immature hematopoiesis is largely elusive. Both direct effects of endocannabinoids on stem cells and indirect effects through endocannabinoid-responsive niche cells like macrophages have been reported. Using two different CB2-deficient mouse models, we studied the role of the endocannabinoid system in immature hematopoiesis. Moreover, we utilized both models to assess the specificity of putative CB2 agonists. As heterodimerization of CB2 and CXCR4, which is highly expressed on hematopoietic stem cells, has already been described, we also assessed potential consequences of CB2 loss for CXCR4/CXCL12 signaling. Overall, no differential effects were observed with any of the compounds tested; the compounds barely induced signaling by themselves, whereas they attenuated CXCL12-induced signals in both CB2-competent and CB2-deficient cells. In vivo experiments were therefore by necessity restricted to loss-of-function studies in knockout (CB2-/-) mice: Except for mild lymphocytosis and slightly elevated circulating progenitor cells, homeostatic hematopoiesis in CB2-/- mice appears to be entirely normal. Mobilization in response to pharmacological stimuli, Plerixafor or G-CSF, was equally potent in wild-type and CB2-/- mice. CB2-/- bone marrow cells reconstituted hematopoiesis in lethally irradiated recipients with engraftment kinetics indistinguishable from those of wild-type grafts. In summary, we found the endocannabinoid system to be largely dispensable for normal murine hematopoiesis.


Asunto(s)
Endocannabinoides/metabolismo , Regulación de la Expresión Génica , Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Modelos Biológicos , Receptor Cannabinoide CB2/biosíntesis , Animales , Endocannabinoides/genética , Células Madre Hematopoyéticas/citología , Ratones , Ratones Noqueados , Receptor Cannabinoide CB2/genética , Receptores CXCR4/genética , Receptores CXCR4/metabolismo
4.
Stem Cells Dev ; 24(6): 737-46, 2015 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-25316534

RESUMEN

Hematopoietic stem and progenitor cells (HSPCs) reside in bone marrow (BM) in an environment rich in CXCL12, the ligand for CXCR4, which is constitutively expressed on all immature hematopoietic cells in BM. This ligand-receptor pair critically controls HSPC retention and (relative) quiescence in BM. Interestingly, in a chemokine-abundant environment, CXCR4 surface expression and CXCL12 sensitivity of BM-residing HSPCs are continuously maintained. The mechanisms underlying this peculiar pattern of G-protein signal integration by BM-HSPCs are unknown. G-protein receptor kinases (GRKs) control receptor function by phosphorylating the intracellular domains upon ligand-induced activation, which results in receptor internalization and transient refractoriness. Using, therefore, a GRK6-deficient (GRK6(-/-)) mouse, we sought to address how perturbed ligand-induced CXCR4 (in)activation affects HSPC behavior in vitro and in vivo. In vitro, GRK6(-/-) HSPCs were characterized by hyper-responsiveness to CXCL12, as expected. In vivo, GRK6(-/-) immature hematopoiesis was characterized by a marked expansion of immature hematopoiesis in spleens and a modest repopulation defect in serial competitive transplantation. Enforced mobilization with granulocyte colony-stimulating factor (G-CSF) and AMD3100 was normal, as was hematopoietic regeneration after noncompetitive transplantation or pharmacological myelosuppression. These observations illustrate that GRK-mediated restriction of CXCR4 signal input after ligand engagement is largely dispensable for BM-resident HSPCs, which may explain how continuous CXCL12 responsiveness of BM-HSPCs can be maintained.


Asunto(s)
Quimiocina CXCL12/metabolismo , Quinasas de Receptores Acoplados a Proteína-G/metabolismo , Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Transducción de Señal , Animales , Células Cultivadas , Quinasas de Receptores Acoplados a Proteína-G/genética , Células Madre Hematopoyéticas/citología , Ratones , Factor Plaquetario 4/metabolismo
5.
Cell Rep ; 11(5): 737-47, 2015 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-25921529

RESUMEN

CXCL12-CXCR4 signaling controls multiple physiological processes and its dysregulation is associated with cancers and inflammatory diseases. To discover as-yet-unknown endogenous ligands of CXCR4, we screened a blood-derived peptide library for inhibitors of CXCR4-tropic HIV-1 strains. This approach identified a 16 amino acid fragment of serum albumin as an effective and highly specific CXCR4 antagonist. The endogenous peptide, termed EPI-X4, is evolutionarily conserved and generated from the highly abundant albumin precursor by pH-regulated proteases. EPI-X4 forms an unusual lasso-like structure and antagonizes CXCL12-induced tumor cell migration, mobilizes stem cells, and suppresses inflammatory responses in mice. Furthermore, the peptide is abundant in the urine of patients with inflammatory kidney diseases and may serve as a biomarker. Our results identify EPI-X4 as a key regulator of CXCR4 signaling and introduce proteolysis of an abundant precursor protein as an alternative concept for chemokine receptor regulation.


Asunto(s)
Fragmentos de Péptidos/metabolismo , Péptidos/metabolismo , Receptores CXCR4/antagonistas & inhibidores , Albúmina Sérica/metabolismo , Secuencia de Aminoácidos , Animales , Biomarcadores/orina , Línea Celular , Movimiento Celular/efectos de los fármacos , Células HEK293 , VIH-1/fisiología , Semivida , Humanos , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Biblioteca de Péptidos , Péptidos/química , Péptidos/farmacología , Unión Proteica , Estructura Terciaria de Proteína , Receptores CXCR4/metabolismo , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Alineación de Secuencia , Albúmina Sérica/química , Albúmina Sérica/farmacología , Transducción de Señal/efectos de los fármacos , Internalización del Virus/efectos de los fármacos
6.
Exp Hematol ; 37(3): 402-15.e1, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19157683

RESUMEN

OBJECTIVE: The CXCR4 antagonist AMD3100 mobilizes hematopoietic stem/progenitor cells (HSPC) in several species. Few data are available on the biology of HSPC mobilized with AMD3100 as single agent. To further study the kinetics and properties of AMD3100-mobilized HSPC, and to explore the size of mobilizable pools of HSPC targeted by AMD3100, we studied the effect of a continuous infusion scheme with saturating doses of AMD3100 [AMDi]. MATERIALS AND METHODS: Using established procedures, we evaluated mice mobilized with AMD3100, or those transplanted with AMD3100-mobilized HSPC. RESULTS: Relative to single-bolus AMD3100 [AMDb], the number of circulating CFU-C or CRU was dramatically higher after [AMDi]. During [AMDi], circulating CFU-C accumulated slowly, but after its discontinuation, CFU-C disappeared rapidly. Compared to bone marrow (BM)-c-kit(+) cells, AMD3100-mobilized (AMDb or AMDi) c-kit(+) cells showed reduced expression of several cytoadhesion molecules, similar to granulocyte colony-stimulating factor-mobilized c-kit(+) cells. In contrast to the latter, expression of CXCR4 and CD26 were not reduced on AMD3100-mobilized c-kit(+) cells. BM homing of [AMDi]-mobilized CFU-C was >50% increased over normal BM-CFU-C. Hematopoietic recovery after transplantation of [AMDi]-mobilized peripheral blood was comparable to that of continuous infusion granulocyte colony-stimulating factor-mobilized peripheral blood. AMD3100-mobilized HSPC were predominantly in G(0), and partial bromodeoxyuridine-labeling experiments documented underrepresentation of labeled cells (<5%) among [AMDb]-mobilized c-kit(+) cells, suggesting that cycling cells in BM, or those that recently completed cell cycle, are not targeted for mobilization by AMD3100. CONCLUSIONS: Our data demonstrate that [AMDi] is an efficacious mobilization scheme fully supporting transplantation demands and expands previous knowledge about properties and size of AMD3100-sensitive BM-HSPC pools.


Asunto(s)
Movilización de Célula Madre Hematopoyética/métodos , Células Madre Hematopoyéticas/efectos de los fármacos , Compuestos Heterocíclicos/farmacología , Animales , Bencilaminas , Células de la Médula Ósea , Recuento de Células , Movimiento Celular , Ciclamas , Células Madre Hematopoyéticas/citología , Compuestos Heterocíclicos/administración & dosificación , Ratones , Trasplante de Células Madre de Sangre Periférica , Receptores CXCR4/antagonistas & inhibidores
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