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1.
Proc Natl Acad Sci U S A ; 120(7): e2208509120, 2023 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-36745791

RESUMO

Antigenic peptides derived from introns are presented on major histocompatibility (MHC) class I molecules, but how these peptides are produced is poorly understood. Here, we show that an MHC class I epitope (SL8) sequence inserted in the second intron of the ß-globin gene in a C57BL/6 mouse (HBB) generates immune tolerance. Introduction of SL8-specific CD8+ T cells derived from OT-1 transgenic mice resulted in a threefold increase in OT-1 T cell proliferation in HBB animals, as compared to wild-type animals. The growth of MCA sarcoma cells expressing the intron-derived SL8 epitope was suppressed in wild-type animals compared to HBB mice. The ß-globin pre-mRNA was detected in the light polysomal fraction, and introducing stop codons identified a non-AUG initiation site between +228 and +255 nts upstream of the SL8. Isolation of ribosome footprints confirmed translation initiation within this 27 nt sequence. Furthermore, treatment with splicing inhibitor shifts the translation of the pre-mRNA to monosomal fractions and results in an increase of intron-derived peptide substrate as shown by polysome profiling and cell imaging. These results show that non-AUG-initiated translation of pre-mRNAs generates peptides for MHC class I immune tolerance and helps explain why alternative tissue-specific splicing is tolerated by the immune system.


Assuntos
Antígenos de Histocompatibilidade Classe I , Precursores de RNA , Animais , Camundongos , Antígenos de Histocompatibilidade Classe I/genética , Precursores de RNA/genética , Linfócitos T CD8-Positivos , Biossíntese de Proteínas , Apresentação de Antígeno , Camundongos Endogâmicos C57BL , Peptídeos/metabolismo , Tolerância Imunológica/genética , Epitopos , Antígenos de Histocompatibilidade Classe II/genética
3.
Nat Commun ; 13(1): 5726, 2022 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-36175410

RESUMO

Membrane-less organelles are condensates formed by phase separation whose functions often remain enigmatic. Upon oxidative stress, PML scaffolds Nuclear Bodies (NBs) to regulate senescence or metabolic adaptation. PML NBs recruit many partner proteins, but the actual biochemical mechanism underlying their pleiotropic functions remains elusive. Similarly, PML role in embryonic stem cell (ESC) and retro-element biology is unsettled. Here we demonstrate that PML is essential for oxidative stress-driven partner SUMO2/3 conjugation in mouse ESCs (mESCs) or leukemia, a process often followed by their poly-ubiquitination and degradation. Functionally, PML is required for stress responses in mESCs. Differential proteomics unravel the KAP1 complex as a PML NB-dependent SUMO2-target in arsenic-treated APL mice or mESCs. PML-driven KAP1 sumoylation enables activation of this key epigenetic repressor implicated in retro-element silencing. Accordingly, Pml-/- mESCs re-express transposable elements and display 2-Cell-Like features, the latter enforced by PML-controlled SUMO2-conjugation of DPPA2. Thus, PML orchestrates mESC state by coordinating SUMO2-conjugation of different transcriptional regulators, raising new hypotheses about PML roles in cancer.


Assuntos
Arsênio , Sumoilação , Animais , Elementos de DNA Transponíveis , Células-Tronco Embrionárias , Camundongos , Corpos Nucleares , Fatores de Transcrição
4.
Int J Mol Sci ; 22(19)2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34638998

RESUMO

During transformation, myelodysplastic syndromes (MDS) are characterized by reducing apoptosis of bone marrow (BM) precursors. Mouse models of high risk (HR)-MDS and acute myelogenous leukemia (AML) post-MDS using mutant NRAS and overexpression of human BCL-2, known to be poor prognostic indicators of the human diseases, were created. We have reported the efficacy of the BCL-2 inhibitor, ABT-737, on the AML post-MDS model; here, we report that this BCL-2 inhibitor also significantly extended survival of the HR-MDS mouse model, with reductions of BM blasts and lineage negative/Sca1+/KIT+ (LSK) cells. Secondary transplants showed increased survival in treated compared to untreated mice. Unlike the AML model, BCL-2 expression and RAS activity decreased following treatment and the RAS:BCL-2 complex remained in the plasma membrane. Exon-specific gene expression profiling (GEP) of HR-MDS mice showed 1952 differentially regulated genes upon treatment, including genes important for the regulation of stem cells, differentiation, proliferation, oxidative phosphorylation, mitochondrial function, and apoptosis; relevant in human disease. Spliceosome genes, found to be abnormal in MDS patients and downregulated in our HR-MDS model, such as Rsrc1 and Wbp4, were upregulated by the treatment, as were genes involved in epigenetic regulation, such as DNMT3A and B, upregulated upon disease progression and downregulated upon treatment.


Assuntos
Compostos de Bifenilo/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/metabolismo , Nitrofenóis/administração & dosagem , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/metabolismo , Sulfonamidas/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Medula Óssea/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Estimativa de Kaplan-Meier , Camundongos , Camundongos Transgênicos , Proteínas Monoméricas de Ligação ao GTP/genética , Síndromes Mielodisplásicas/mortalidade , Piperazinas/administração & dosagem , Proteínas Proto-Oncogênicas c-bcl-2/genética , Células-Tronco/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos
5.
Oncoimmunology ; 9(1): 1728871, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32158621

RESUMO

Despite considerable progress, the treatment of acute leukemia continues to be a challenge for a significant majority of patients. Using a well-characterized preclinical mouse model of acute promyelocytic leukemia (APL), we evaluated here the antileukemic efficacy of RT53, an anticancer peptide with potential immunological properties. Our results indicate that RT53 possesses a direct antileukemic effect, even at a late stage. We also demonstrate that a single injection of a vaccine consisting of leukemic blasts exposed to RT53, which induces the hallmarks of immunogenic cell death, was highly effective in preventing leukemia development in both prophylactic and therapeutic settings. The vaccine comprising RT53-treated APL cells generated long-term antileukemic protection and depletion experiments indicated that CD4 + T cells were of crucial importance for vaccine efficacy. Combined, our results provide the rationale for the exploration of RT53-based therapies for the treatment of acute leukemia.


Assuntos
Leucemia Mieloide Aguda , Leucemia Promielocítica Aguda , Animais , Humanos , Camundongos , Peptídeos , Linfócitos T
6.
Nucleic Acids Res ; 47(6): 3086-3100, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30624716

RESUMO

Peptides presented on major histocompatibility (MHC) class I molecules form an essential part of the immune system's capacity to detect virus-infected or transformed cells. Earlier works have shown that pioneer translation peptides (PTPs) for the MHC class I pathway are as efficiently produced from introns as from exons, or from mRNAs targeted for the nonsense-mediated decay pathway. The production of PTPs is a target for viral immune evasion but the underlying molecular mechanisms that govern this non-canonical translation are unknown. Here, we have used different approaches to show how events taking place on the nascent transcript control the synthesis of PTPs and full-length proteins. By controlling the subcellular interaction between the G-quadruplex structure (G4) of a gly-ala encoding mRNA and nucleolin (NCL) and by interfering with mRNA maturation using multiple approaches, we demonstrate that antigenic peptides derive from a nuclear non-canonical translation event that is independently regulated from the synthesis of full-length proteins. Moreover, we show that G4 are exploited to control mRNA localization and translation by distinguishable mechanisms that are targets for viral immune evasion.


Assuntos
Antígenos/genética , Antígenos de Histocompatibilidade Classe I/genética , Peptídeos/genética , Biossíntese de Proteínas/genética , Antígenos/imunologia , Núcleo Celular/genética , Núcleo Celular/imunologia , Quadruplex G , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Evasão da Resposta Imune/genética , Evasão da Resposta Imune/imunologia , Degradação do RNAm Mediada por Códon sem Sentido/genética , Degradação do RNAm Mediada por Códon sem Sentido/imunologia , Peptídeos/imunologia , Biossíntese de Proteínas/imunologia , RNA Mensageiro/genética , RNA Mensageiro/imunologia
7.
Stem Cell Reports ; 11(5): 1075-1091, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30449320

RESUMO

Fanconi anemia (FA) causes bone marrow failure early during childhood, and recent studies indicate that a hematopoietic defect could begin in utero. We performed a unique kinetics study of hematopoiesis in Fancg-/- mouse embryos, between the early embryonic day 11.5 (E11.5) to E12.5 developmental window (when the highest level of hematopoietic stem cells [HSC] amplification takes place) and E14.5. This study reveals a deep HSC defect with exhaustion of proliferative and self-renewal capacities very early during development, together with severe FA clinical and biological manifestations, which are mitigated at E14.5 due to compensatory mechanisms that help to ensure survival of Fancg-/- embryos. It also reports that a deep HSC defect is also observed during human FA development, and that human FA fetal liver (FL) HSCs present a transcriptome profile similar to that of mouse E12.5 Fancg-/- FL HSCs. Altogether, our results highlight that early mouse FL could represent a good alternative model for studying Fanconi pathology.


Assuntos
Desenvolvimento Embrionário , Anemia de Fanconi/patologia , Células-Tronco Hematopoéticas/patologia , Animais , Apoptose , Ciclo Celular , Dano ao DNA , Embrião de Mamíferos/patologia , Eritrócitos/metabolismo , Proteína do Grupo de Complementação G da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação G da Anemia de Fanconi/metabolismo , Feminino , Ontologia Genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Fígado/embriologia , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Fenótipo , Placenta/metabolismo , Gravidez , Transcriptoma/genética
8.
Cancer Discov ; 8(12): 1614-1631, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30266814

RESUMO

Deletion of chromosome 6q is a well-recognized abnormality found in poor-prognosis T-cell acute lymphoblastic leukemia (T-ALL). Using integrated genomic approaches, we identified two candidate haploinsufficient genes contiguous at 6q14, SYNCRIP (encoding hnRNP-Q) and SNHG5 (that hosts snoRNAs), both involved in regulating RNA maturation and translation. Combined silencing of both genes, but not of either gene alone, accelerated leukemogeneis in a Tal1/Lmo1/Notch1-driven mouse model, demonstrating the tumor-suppressive nature of the two-gene region. Proteomic and translational profiling of cells in which we engineered a short 6q deletion by CRISPR/Cas9 genome editing indicated decreased ribosome and mitochondrial activities, suggesting that the resulting metabolic changes may regulate tumor progression. Indeed, xenograft experiments showed an increased leukemia-initiating cell activity of primary human leukemic cells upon coextinction of SYNCRIP and SNHG5. Our findings not only elucidate the nature of 6q deletion but also highlight the role of ribosomes and mitochondria in T-ALL tumor progression. SIGNIFICANCE: The oncogenic role of 6q deletion in T-ALL has remained elusive since this chromosomal abnormality was first identified more than 40 years ago. We combined genomic analysis and functional models to show that the codeletion of two contiguous genes at 6q14 enhances malignancy through deregulation of a ribosome-mitochondria axis, suggesting the potential for therapeutic intervention.This article is highlighted in the In This Issue feature, p. 1494.


Assuntos
Deleção Cromossômica , Ribonucleoproteínas Nucleares Heterogêneas/genética , Leucemia de Células T/genética , RNA Longo não Codificante/genética , Ribossomos/metabolismo , Animais , Linhagem Celular Tumoral , Cromossomos Humanos Par 6 , Progressão da Doença , Perfilação da Expressão Gênica , Haploinsuficiência , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Leucemia de Células T/metabolismo , Leucemia de Células T/patologia , Camundongos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Interferência de RNA , RNA Longo não Codificante/metabolismo , Transplante Heterólogo
9.
Stem Cell Reports ; 6(6): 970-984, 2016 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-27304919

RESUMO

The capacity of hematopoietic stem cells (HSC) to generate B lymphocytes declines with age, contributing to impaired immune function in the elderly. Here we show that the histone methyltransferase SUV39H1 plays an important role in human B lymphoid differentiation and that expression of SUV39H1 decreases with age in both human and mouse HSC, leading to a global reduction in H3K9 trimethylation and perturbed heterochromatin function. Further, we demonstrate that SUV39H1 is a target of microRNA miR-125b, a known regulator of HSC function, and that expression of miR-125b increases with age in human HSC. Overexpression of miR-125b and inhibition of SUV39H1 in young HSC induced loss of B cell potential. Conversely, both inhibition of miR-125 and enforced expression of SUV39H1 improved the capacity of HSC from elderly individuals to generate B cells. Our findings highlight the importance of heterochromatin regulation in HSC aging and B lymphopoiesis.


Assuntos
Envelhecimento/imunologia , Linfócitos B/citologia , Células-Tronco Hematopoéticas/citologia , Linfopoese/imunologia , Metiltransferases/genética , MicroRNAs/genética , Proteínas Repressoras/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Animais , Antagomirs/genética , Antagomirs/metabolismo , Linfócitos B/imunologia , Sequência de Bases , Diferenciação Celular , Feminino , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/imunologia , Heterocromatina/química , Heterocromatina/metabolismo , Humanos , Linfopoese/genética , Masculino , Metiltransferases/imunologia , Camundongos , MicroRNAs/antagonistas & inibidores , MicroRNAs/imunologia , Cultura Primária de Células , Proteínas Repressoras/imunologia , Transdução de Sinais
10.
J Hematol Oncol ; 9: 5, 2016 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-26817437

RESUMO

BACKGROUND: In spite of the recent discovery of genetic mutations in most myelodysplasic (MDS) patients, the pathophysiology of these disorders still remains poorly understood, and only few in vivo models are available to help unravel the disease. METHODS: We performed global specific gene expression profiling and functional pathway analysis in purified Sca1+ cells of two MDS transgenic mouse models that mimic human high-risk MDS (HR-MDS) and acute myeloid leukemia (AML) post MDS, with NRASD12 and BCL2 transgenes under the control of different promoters MRP8NRASD12/tethBCL-2 or MRP8[NRASD12/hBCL-2], respectively. RESULTS: Analysis of dysregulated genes that were unique to the diseased HR-MDS and AML post MDS mice and not their founder mice pointed first to pathways that had previously been reported in MDS patients, including DNA replication/damage/repair, cell cycle, apoptosis, immune responses, and canonical Wnt pathways, further validating these models at the gene expression level. Interestingly, pathways not previously reported in MDS were discovered. These included dysregulated genes of noncanonical Wnt pathways and energy and lipid metabolisms. These dysregulated genes were not only confirmed in a different independent set of BM and spleen Sca1+ cells from the MDS mice but also in MDS CD34+ BM patient samples. CONCLUSIONS: These two MDS models may thus provide useful preclinical models to target pathways previously identified in MDS patients and to unravel novel pathways highlighted by this study.


Assuntos
Perfilação da Expressão Gênica/métodos , Leucemia Mieloide/genética , Síndromes Mielodisplásicas/genética , Transdução de Sinais/genética , Doença Aguda , Animais , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Mieloide/patologia , Camundongos , Camundongos Transgênicos , Síndromes Mielodisplásicas/patologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco
11.
Oncotarget ; 6(32): 32494-508, 2015 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-26378812

RESUMO

We have previously shown that a specific promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) DNA vaccine combined with all-trans retinoic acid (ATRA) increases the number of long term survivors with enhanced immune responses in a mouse model of acute promyelocytic leukemia (APL). This study reports the efficacy of a non-specific DNA vaccine, pVAX14Flipper (pVAX14), in both APL and high risk myelodysplastic syndrome (HR-MDS) models. PVAX14 is comprised of novel immunogenic DNA sequences inserted into the pVAX1 therapeutic plasmid. APL mice treated with pVAX14 combined with ATRA had increased survival comparable to that obtained with a specific PML-RARA vaccine. Moreover, the survival advantage correlated with decreased PML-RARA transcript levels and increase in anti-RARA antibody production. In HR-MDS mice, pVAX14 significantly improved survival and reduced biomarkers of leukemic transformation such as phosphorylated mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1. In both preclinical models, pVAX14 vaccine significantly increased interferon gamma (IFNγ) production, memory T-cells (memT), reduced the number of colony forming units (CFU) and increased expression of the adapter molecule signalling to NF-κB, MyD88. These results demonstrate the adjuvant properties of pVAX14 providing thus new approaches to improve clinical outcome in two different models of myeloid malignancies, which may have potential for a broader applicability in other cancers.


Assuntos
Adjuvantes Imunológicos/farmacologia , Vacinas Anticâncer/farmacologia , Leucemia Promielocítica Aguda/tratamento farmacológico , Síndromes Mielodisplásicas/tratamento farmacológico , Neoplasias Experimentais/tratamento farmacológico , Tretinoína/farmacologia , Vacinas de DNA/farmacologia , Animais , Anticorpos/sangue , Sequência de Bases , Vacinas Anticâncer/imunologia , Regulação Neoplásica da Expressão Gênica , Genes ras , Memória Imunológica/efeitos dos fármacos , Interferon gama/imunologia , Interferon gama/metabolismo , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/imunologia , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Camundongos Transgênicos , Dados de Sequência Molecular , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/imunologia , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Tempo , Carga Tumoral/efeitos dos fármacos , Vacinação , Vacinas de DNA/imunologia
12.
Blood ; 122(16): 2864-76, 2013 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-23943652

RESUMO

Myelodysplastic syndrome (MDS) transforms into an acute myelogenous leukemia (AML) with associated increased bone marrow (BM) blast infiltration. Using a transgenic mouse model, MRP8[NRASD12/hBCL-2], in which the NRAS:BCL-2 complex at the mitochondria induces MDS progressing to AML with dysplastic features, we studied the therapeutic potential of a BCL-2 homology domain 3 mimetic inhibitor, ABT-737. Treatment significantly extended lifespan, increased survival of lethally irradiated secondary recipients transplanted with cells from treated mice compared with cells from untreated mice, with a reduction of BM blasts, Lin-/Sca-1(+)/c-Kit(+), and progenitor populations by increased apoptosis of infiltrating blasts of diseased mice assessed in vivo by technicium-labeled annexin V single photon emission computed tomography and ex vivo by annexin V/7-amino actinomycin D flow cytometry, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, caspase 3 cleavage, and re-localization of the NRAS:BCL-2 complex from mitochondria to plasma membrane. Phosphoprotein analysis showed restoration of wild-type (WT) AKT or protein kinase B, extracellular signal-regulated kinase 1/2 and mitogen-activated protein kinase patterns in spleen cells after treatment, which showed reduced mitochondrial membrane potential. Exon specific gene expression profiling corroborates the reduction of leukemic cells, with an increase in expression of genes coding for stem cell development and maintenance, myeloid differentiation, and apoptosis. Myelodysplastic features persist underscoring targeting of BCL-2-mediated effects on MDS-AML transformation and survival of leukemic cells.


Assuntos
Compostos de Bifenilo/farmacologia , Leucemia Mieloide Aguda/metabolismo , Nitrofenóis/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/farmacologia , Proteínas ras/metabolismo , Animais , Antígenos Ly/metabolismo , Linhagem da Célula , Membrana Celular/metabolismo , Proliferação de Células , Transformação Celular Neoplásica , Transplante de Células , Modelos Animais de Doenças , Citometria de Fluxo , Regulação Leucêmica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/metabolismo , Células-Tronco/citologia
13.
J Immunol ; 190(12): 6187-97, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23690469

RESUMO

In this study, we identify transmembrane protein 131-like (TMEM131L) as a novel regulator of thymocyte proliferation and demonstrate that it corresponds to a not as yet reported inhibitor of Wnt signaling. Short hairpin RNA-mediated silencing of TMEM131L in human CD34(+) hematopoietic progenitors, which were then grafted in NOD-SCID/IL-2rγ(null) mice, resulted in both thymocyte hyperproliferation and multiple pre- and post-ß-selection intrathymic developmental defects. Consistent with deregulated Wnt signaling, TMEM131L-deficient thymocytes expressed Wnt target genes at abnormally high levels, and they displayed both constitutive phosphorylation of Wnt coreceptor LRP6 and ß-catenin intranuclear accumulation. Using T cell factor reporter assays, we found that membrane-associated TMEM131L inhibited canonical Wnt/ß-catenin signaling at the LRP6 coreceptor level. Whereas membrane-associated TMEM131L did not affect LRP6 expression under basal conditions, it triggered lysosome-dependent degradation of its active phosphorylated form following Wnt activation. Genetic mapping showed that phosphorylated LRP6 degradation did not depend on TMEM131L cytoplasmic part but rather on a conserved extracellular domain proximal to the membrane. Collectively, these data indicate that, during thymopoiesis, stage-specific surface translocation of TMEM131L may regulate immature single-positive thymocyte proliferation arrest by acting through mixed Wnt-dependent and -independent mechanisms.


Assuntos
Proliferação de Células , Proteínas de Membrana/metabolismo , Timócitos/citologia , Via de Sinalização Wnt/fisiologia , Animais , Citometria de Fluxo , Células HEK293 , Humanos , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Microscopia de Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timócitos/imunologia
14.
Mol Cell Probes ; 27(1): 1-5, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22906630

RESUMO

Using an acute promyelocytic leukemia (APL) preclinical model, we show that oncogene-specific PCR (Polymerase Chain Reaction)-based assays allow to evaluate the efficacy of immunotherapy combining all-trans retinoic acid (ATRA) and a DNA-based vaccine targeting the promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) oncogene. Kaplan-Meier survival analysis according to the peripheral blood PML-RARα normalized copy number (NCN) clearly shows that ATRA + DNA-treated mice with an NCN lower than 10 (43%) formed the group with a highly significant (p < 0.0001) survival advantage. Furthermore, a PCR assay was used to assess various tissues and organs for the presence of PML-RARα-positive cells in long-term survivors (n = 15). As expected, the majority of mice (n = 10) had no measurable tissue level of PML-RARα. However, five mice showed a weak positive signal in both the brain and spleen (n = 2), in the brain only (n = 2) and in the spleen only (n = 1). Thus tracking the oncogene-positive cells in long-term survivors reveals for the first time that extramedullary PML-RARα-positive cell reservoirs such as the brain may persist and be involved in relapses.


Assuntos
Imunoterapia , Leucemia Promielocítica Aguda/terapia , Proteínas de Fusão Oncogênica/metabolismo , Tretinoína/uso terapêutico , Vacinas de DNA/uso terapêutico , Animais , Encéfalo/citologia , Dosagem de Genes , Estimativa de Kaplan-Meier , Leucemia Promielocítica Aguda/mortalidade , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/uso terapêutico , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/imunologia , Baço/citologia , Resultado do Tratamento
15.
Leuk Res ; 37(3): 312-9, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23153525

RESUMO

We have previously demonstrated that two prognostic features of myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML), mutant NRAS and over-expressing BCL-2, cooperate physically and functionally in vivo. Screening of MDS patient bone marrow (BM) identified NRAS:BCL-2 co-localization in 64% cases, correlating with percentage BM blasts, apoptotic features and disease status (p<0.0001). Localization of the complex at the plasma membrane or the mitochondria correlated with disease and apoptosis features in MDS patients, whilst caspase-9 mediated mechanism was elucidated in vivo and in vitro. The intensity and localization of the RAS:BCL-2 complex merits further evaluation as a novel biomarker of MDS.


Assuntos
Apoptose , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas ras/metabolismo , Animais , Apoptose/genética , Membrana Celular/metabolismo , Progressão da Doença , Genes ras , Humanos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Síndromes Mielodisplásicas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Distribuição Tecidual/fisiologia
16.
J Immunol ; 189(4): 1648-60, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22798679

RESUMO

To model the developmental pattern of human prothymocytes and thymopoiesis, we used NOD-scid/γc(-/-) mice grafted with human umbilical cord blood CD34(+) hematopoietic progenitor cells (HPCs). Human prothymocytes developed in the murine bone marrow (BM) from multipotent CD34(++)CD38(lo)lineage(-) HPCs to CD34(++)CD7(+)CD2(-) pro-T1 cells that progressed in a Notch-dependent manner to CD34(+)CD7(++)CD2(+) pro-T2 cells, which migrated to the thymus. BM prothymocyte numbers peaked 1 mo after graft, dropped at mo 2, and persisted at low levels thereafter, with only a few CD34(+)CD7(lo) prothymocytes with limited T potential being detected by mo 5. As a consequence, thymopoiesis in this xenogeneic setting began by weeks 4-6, peaked at mo 3, and decreased thenceforth. Analyzing mice grafted at 2, 4 or 8, mo of age showed that in an "older" BM, prothymocyte differentiation was perturbed and resulted in CD34(+)CD7(lo) prothymocytes with limited T potential. Whereas the early drop in BM thymopoietic activity was related to a Notch-independent loss of T potential by CD34(++)CD38(lo)lineage(-) HPCs, the later age-dependent production decline of prothymocytes was linked to a more complex mix of cell-intrinsic and microenvironmental defects. Accordingly, and contrasting with what was observed with umbilical cord blood HPCs, CD34(+) HPCs from human adult BM displayed only marginal thymopoietic activity when grafted into young 2-mo-old NOD-scid/γc(-/-) mice. These data demonstrate that the developmental pattern of BM prothymocytes during human late fetal and early postnatal life can be reproduced in humanized mice, and they suggest that onset of human thymus involution relates to decreased colonization by prothymocytes.


Assuntos
Diferenciação Celular/imunologia , Células Progenitoras Linfoides/citologia , Linfopoese/fisiologia , Linfócitos T/citologia , Timo/citologia , Animais , Células da Medula Óssea/citologia , Linhagem da Célula/imunologia , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo
17.
Cell Stem Cell ; 11(1): 36-49, 2012 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-22683204

RESUMO

Fanconi anemia (FA) is an inherited DNA repair deficiency syndrome. FA patients undergo progressive bone marrow failure (BMF) during childhood, which frequently requires allogeneic hematopoietic stem cell transplantation. The pathogenesis of this BMF has been elusive to date. Here we found that FA patients exhibit a profound defect in hematopoietic stem and progenitor cells (HSPCs) that is present before the onset of clinical BMF. In response to replicative stress and unresolved DNA damage, p53 is hyperactivated in FA cells and triggers a late p21(Cdkn1a)-dependent G0/G1 cell-cycle arrest. Knockdown of p53 rescued the HSPC defects observed in several in vitro and in vivo models, including human FA or FA-like cells. Taken together, our results identify an exacerbated p53/p21 "physiological" response to cellular stress and DNA damage accumulation as a central mechanism for progressive HSPC elimination in FA patients, and have implications for clinical care.


Assuntos
Medula Óssea/patologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patologia , Células-Tronco Hematopoéticas/patologia , Proteína Supressora de Tumor p53/metabolismo , Adolescente , Adulto , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/patologia , Envelhecimento/patologia , Animais , Medula Óssea/metabolismo , Criança , Pré-Escolar , Modelos Animais de Doenças , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/patologia , Proteína do Grupo de Complementação C da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação C da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular , Pontos de Checagem da Fase G2 do Ciclo Celular , Técnicas de Silenciamento de Genes , Inativação Gênica , Células-Tronco Hematopoéticas/metabolismo , Humanos , Lactente , Camundongos , Pessoa de Meia-Idade , Fase S
18.
Blood ; 118(7): 1784-96, 2011 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-21715312

RESUMO

The mechanisms regulating the emergence of BM prothymocytes remain poorly characterized. Genome-wide transcriptome analyses looking for genes expressed in human prothymocytes led to the identification of AF1q/MLLT11 as a candidate gene conceivably involved in this process. Analysis of AF1q protein subcellular localization and intracellular trafficking showed that despite pronounced karyophily, it was subjected to constitutive nuclear export followed by ubiquitin-mediated degradation in the centrosomal area. Using in vitro assays based on either forced expression or shRNA-mediated silencing of AF1q, we provide evidence that the protein promotes T- over B-cell differentiation in multipotent hematopoietic progenitors. At the molecular level, AF1q confers to multipotent progenitors an increased susceptibility to Delta-like/Notch-mediated signaling. Consistent with these findings, enforced AF1q expression in humanized mice fosters the emergence of BM CD34(+)CD7(+) prothymocytes, enhances subsequent thymus colonization, and accelerates intrathymic T-cell development. In contrast, AF1q silencing provokes a global shift of BM lymphopoiesis toward the B-cell lineage, hinders prothymocyte development, inhibits thymus colonization, and leads to intrathymic accumulation of B cells. Our results indicate that AF1q cooperates with the Notch signaling pathway to foster the emergence of BM prothymocytes and drive subsequent intrathymic specification toward the T-cell lineage.


Assuntos
Células-Tronco Hematopoéticas/citologia , Linfopoese , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Notch/metabolismo , Linfócitos T/citologia , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Células Cultivadas , Inativação Gênica , Células HeLa , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Camundongos SCID , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas/genética , Alinhamento de Sequência , Transdução de Sinais , Linfócitos T/metabolismo
19.
Blood ; 115(3): 653-6, 2010 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-19965687

RESUMO

DNA vaccination and all-trans retinoic acid (ATRA) result in a survival advantage in a mouse model of acute promyelocytic leukemia (APL). Depletion of CD4(+) or CD8(+) cells abolished this effect. CD4(+) depletions of long-term survivors resulted in relapse and death within 3 months, thus demonstrating the need of both CD4(+) and CD8(+) subsets for the generation of DNA-driven antileukemic immune responses and underscoring a crucial role of CD4(+) cells in the maintenance of durable remissions. Degranulation and cytotoxic carboxyfluorescein diacetate succinimidyl ester-based assays showed major histocompatibility complex-restricted APL-specific T cell-mediated immune responses. Sorted APL-specific CD8(+)CD107a(+) T cells showed an increase of antileukemic activity. Effectors from ATRA + DNA-treated mice were shown to secrete interferon-gamma when stimulated with either APL cells or peptides from the promyelocytic leukemia-RARalpha vaccine-derived sequences as detected by ELISpot assays. Our results demonstrate that DNA vaccination with ATRA confers the effective boosting of interferon-gamma-producing and cytotoxic T cells in the leukemic mice.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunidade Celular , Leucemia Promielocítica Aguda/terapia , Tretinoína/administração & dosagem , Vacinas de DNA/administração & dosagem , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Terapia Combinada , Modelos Animais de Doenças , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/genética , Leucemia Promielocítica Aguda/imunologia , Leucemia Promielocítica Aguda/patologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Camundongos , Proteínas de Fusão Oncogênica/administração & dosagem , Proteínas de Fusão Oncogênica/genética , Análise de Sobrevida , Resultado do Tratamento , Células Tumorais Cultivadas , Vacinas de DNA/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Ophthalmic Res ; 40(3-4): 175-80, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18421235

RESUMO

PURPOSE: A major increased risk of developing birdshot chorioretinopathy is reported in humans who are HLA-A29-positive. To better characterize this disease, an animal model of HLA-A29-associated disease was developed and the pathology arising spontaneously in these transgenic mice was compared to animal models of autoimmune uveoretinitis and to human pathology. MATERIALS AND METHODS: HLA-A2902 cDNA (A29c) was obtained from a patient suffering from birdshot retinochoroidopathy and used for transgene construct to generate HLA-A29 transgenic mice. Histopathological examination of the animal cohort was performed up to 15 months of age. It was compared with the ocular pathology developed in C57BL/6 mice and in Lewis rats immunized with retinal autoantigens. RESULTS: Aging HLA-A29 transgenic mice spontaneously developed an ocular disease with resemblance to experimental retinal-Ag-induced autoimmune ocular disease and to human pathologies shown in birdshot retinochoroidopathy, Vogt-Koyanagi-Harada and sympathetic ophthalmia. Pathogenic mechanisms could possibly be shared by these conditions. CONCLUSION: Humanized models of ocular inflammation developed in HLA class I and class II transgenic mice will help better understand the mechanisms responsible for ocular inflammation. Local control of autoimmunity in HLA-A29-positive individuals would be an important option for new therapeutic strategies.


Assuntos
Doenças Autoimunes/patologia , Autoimunidade/imunologia , Modelos Animais de Doenças , Retina/imunologia , Uveíte/patologia , Animais , Doenças Autoimunes/imunologia , Progressão da Doença , Feminino , Seguimentos , Antígenos HLA-A/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ratos , Ratos Endogâmicos Lew , Índice de Gravidade de Doença , Uveíte/imunologia , Microglobulina beta-2/imunologia
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