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1.
Blood ; 144(8): 809-821, 2024 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-38875504

RESUMO

ABSTRACT: Epidemiological studies report opposing influences of infection on childhood B-cell acute lymphoblastic leukemia (B-ALL). Although infections in the first year of life appear to exert the largest impact on leukemia risk, the effect of early pathogen exposure on the fetal preleukemia cells (PLC) that lead to B-ALL has yet to be reported. Using cytomegalovirus (CMV) infection as a model early-life infection, we show that virus exposure within 1 week of birth induces profound depletion of transplanted E2A-PBX1 and hyperdiploid B-ALL cells in wild-type recipients and in situ-generated PLC in Eµ-ret mice. The age-dependent depletion of PLC results from an elevated STAT4-mediated cytokine response in neonates, with high levels of interleukin (IL)-12p40-driven interferon (IFN)-γ production inducing PLC death. Similar PLC depletion can be achieved in adult mice by impairing viral clearance. These findings provide mechanistic support for potential inhibitory effects of early-life infection on B-ALL progression and could inform novel therapeutic or preventive strategies.


Assuntos
Modelos Animais de Doenças , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Animais , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Infecções por Citomegalovirus , Pré-Leucemia/genética , Pré-Leucemia/patologia , Camundongos Endogâmicos C57BL , Animais Recém-Nascidos , Diploide
2.
Proc Natl Acad Sci U S A ; 119(51): e2212723119, 2022 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-36508659

RESUMO

The design of selective metal-binding sites is a challenge in both small-molecule and macromolecular chemistry. Selective recognition of manganese (II)-the first-row transition metal ion that tends to bind with the lowest affinity to ligands, as described by the Irving-Williams series-is particularly difficult. As a result, there is a dearth of chemical biology tools with which to study manganese physiology in live cells, which would advance understanding of photosynthesis, host-pathogen interactions, and neurobiology. Here we report the rational re-engineering of the lanthanide-binding protein, lanmodulin, into genetically encoded fluorescent sensors for MnII, MnLaMP1 and MnLaMP2. These sensors with effective Kd(MnII) of 29 and 7 µM, respectively, defy the Irving-Williams series to selectively detect MnII in vitro and in vivo. We apply both sensors to visualize kinetics of bacterial labile manganese pools. Biophysical studies indicate the importance of coordinated solvent and hydrophobic interactions in the sensors' selectivity. Our results establish lanmodulin as a versatile scaffold for design of selective protein-based biosensors and chelators for metals beyond the f-block.


Assuntos
Manganês , Metais , Manganês/metabolismo , Metais/metabolismo , Cinética , Ligantes
3.
Clin Infect Dis ; 76(3): e400-e408, 2023 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35616119

RESUMO

BACKGROUND: The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible in vaccinated and unvaccinated populations. The dynamics that govern its establishment and propensity toward fixation (reaching 100% frequency in the SARS-CoV-2 population) in communities remain unknown. Here, we describe the dynamics of Omicron at 3 institutions of higher education (IHEs) in the greater Boston area. METHODS: We use diagnostic and variant-specifying molecular assays and epidemiological analytical approaches to describe the rapid dominance of Omicron following its introduction into 3 IHEs with asymptomatic surveillance programs. RESULTS: We show that the establishment of Omicron at IHEs precedes that of the state and region and that the time to fixation is shorter at IHEs (9.5-12.5 days) than in the state (14.8 days) or region. We show that the trajectory of Omicron fixation among university employees resembles that of students, with a 2- to 3-day delay. Finally, we compare cycle threshold values in Omicron vs Delta variant cases on college campuses and identify lower viral loads among college affiliates who harbor Omicron infections. CONCLUSIONS: We document the rapid takeover of the Omicron variant at IHEs, reaching near-fixation within the span of 9.5-12.5 days despite lower viral loads, on average, than the previously dominant Delta variant. These findings highlight the transmissibility of Omicron, its propensity to rapidly dominate small populations, and the ability of robust asymptomatic surveillance programs to offer early insights into the dynamics of pathogen arrival and spread.


Assuntos
COVID-19 , Humanos , COVID-19/epidemiologia , SARS-CoV-2/genética , Universidades , Boston
4.
J Labelled Comp Radiopharm ; 66(2): 34-40, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36593743

RESUMO

We report here the detailed radiosynthesis of [18 F]mG4P027, a metabotropic glutamate receptor 4 (mGluR4) PET radiotracer, which showed superior properties to the currently reported mGluR4 radiotracers. The radiosynthesis in the automated system has been challenging, therefore we disclose here the major limiting factors for the synthesis via step-by-step examination. And we hope this thorough study will help its automation for human use in the future.


Assuntos
Compostos Radiofarmacêuticos , Receptores de Glutamato Metabotrópico , Humanos , Tomografia por Emissão de Pósitrons/métodos , Automação , Radioisótopos de Flúor
5.
Angew Chem Int Ed Engl ; 61(3): e202114019, 2022 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-34814231

RESUMO

Fe3+ complexes in aqueous solution can exist as discrete mononuclear species or multinuclear magnetically coupled species. Stimuli-driven change to Fe3+ speciation represents a powerful mechanistic basis for magnetic resonance sensor technology, but ligand design strategies to exert precision control of aqueous Fe3+ magnetostructural properties are entirely underexplored. In pursuit of this objective, we rationally designed a ligand to strongly favor a dinuclear µ-oxo-bridged and antiferromagnetically coupled complex, but which undergoes carboxylesterase mediated transformation to a mononuclear high-spin Fe3+ chelate resulting in substantial T1 -relaxivity increase. The data communicated demonstrate proof of concept for a novel and effective strategy to exert biochemical control over aqueous Fe3+ magnetic, structural, and relaxometric properties.


Assuntos
Carboxilesterase/metabolismo , Compostos Férricos/metabolismo , Compostos Férricos/química , Estrutura Molecular
6.
Genes Dev ; 27(1): 98-115, 2013 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23307870

RESUMO

Transcriptional profiling is a powerful approach for understanding development and disease. Current cell type-specific RNA purification methods have limitations, including cell dissociation trauma or inability to identify all RNA species. Here, we describe "mouse thiouracil (TU) tagging," a genetic and chemical intersectional method for covalent labeling and purification of cell type-specific RNA in vivo. Cre-induced expression of uracil phosphoribosyltransferase (UPRT) provides spatial specificity; injection of 4-thiouracil (4TU) provides temporal specificity. Only UPRT(+) cells exposed to 4TU produce thio-RNA, which is then purified for RNA sequencing (RNA-seq). This method can purify transcripts from spatially complex and rare (<5%) cells, such as Tie2:Cre(+) brain endothelia/microglia (76% validated by expression pattern), or temporally dynamic transcripts, such as those acutely induced by lipopolysaccharide (LPS) injection. Moreover, generating chimeric mice via UPRT(+) bone marrow transplants identifies immune versus niche spleen RNA. TU tagging provides a novel method for identifying actively transcribed genes in specific cells at specific times within intact mice.


Assuntos
Biologia Molecular/métodos , RNA/isolamento & purificação , Coloração e Rotulagem/métodos , Tiouracila/metabolismo , Animais , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Encéfalo/embriologia , Encéfalo/metabolismo , Quimera , Perfilação da Expressão Gênica , Camundongos , Transgenes/genética
7.
Gene Ther ; 27(10-11): 525-534, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-32704085

RESUMO

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have promising potential for opening new avenues in regenerative medicine. However, since the tumorigenic potential of undifferentiated pluripotent stem cells (PSCs) is a major safety concern for clinical transplantation, inducible Caspase-9 (iC9) is under consideration for use as a fail-safe system. Here, we used targeted gene editing to introduce the iC9 system into human iPSCs, and then interrogated the efficiency of inducible apoptosis with normal iPSCs as well as diseased iPSCs derived from patients with acute myeloid leukemia (AML-iPSCs). The iC9 system induced quick and efficient apoptosis to iPSCs in vitro. More importantly, complete eradication of malignant cells without AML recurrence was shown in disease mouse models by using AML-iPSCs. In parallel, it shed light on several limitations of the iC9 system usage. Our results suggest that careful use of the iC9 system will serve as an important countermeasure against posttransplantation adverse events in stem cell transplantation therapies.


Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Animais , Apoptose , Caspase 9/genética , Caspase 9/metabolismo , Diferenciação Celular , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Células-Tronco Pluripotentes/metabolismo
8.
Haematologica ; 104(9): 1744-1755, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30792210

RESUMO

The balance between self-renewal and differentiation is crucial to ensure the homeostasis of the hematopoietic system, and is a hallmark of hematopoietic stem cells. However, the underlying molecular pathways, including the role of micro-RNA, are not completely understood. To assess the contribution of micro-RNA, we performed micro-RNA profiling of hematopoietic stem cells and their immediate downstream progeny multi-potent progenitors from wild-type control and Pbx1-conditional knockout mice, whose stem cells display a profound self-renewal defect. Unsupervised hierarchical cluster analysis separated stem cells from multi-potent progenitors, suggesting that micro-RNA might regulate the first transition step in the adult hematopoietic development. Notably, Pbx1-deficient and wild-type cells clustered separately, linking micro-RNAs to self-renewal impairment. Differential expression analysis of micro-RNA in the physiological stem cell-to-multi-potent progenitor transition and in Pbx1-deficient stem cells compared to control stem cells revealed miR-127-3p as the most differentially expressed. Furthermore, miR-127-3p was strongly stem cell-specific, being quickly down-regulated upon differentiation and not re-expressed further downstream in the bone marrow hematopoietic hierarchy. Inhibition of miR-127-3p function in Lineage-negative cells, achieved through a lentiviral-sponge vector, led to severe stem cell depletion, as assessed with serial transplantation assays. miR-127-3p-sponged stem cells displayed accelerated differentiation, which was uncoupled from proliferation, accounting for the observed stem cell reduction. miR-127-3p overexpression in Lineage-negative cells did not alter stem cell pool size, but gave rise to lymphopenia, likely due to lack of miR-127-3p physiological downregulation beyond the stem cell stage. Thus, tight regulation of miR-127-3p is crucial to preserve the self-renewing stem cell pool and homeostasis of the hematopoietic system.


Assuntos
Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , MicroRNAs/fisiologia , Animais , Linhagem da Célula/genética , Análise por Conglomerados , Cruzamentos Genéticos , Perfilação da Expressão Gênica , Hematopoese , Homeostase , Humanos , Células K562 , Lentivirus/genética , Camundongos , Camundongos Knockout , Estresse Oxidativo , Fator de Transcrição 1 de Leucemia de Células Pré-B/metabolismo
9.
Nucleic Acids Res ; 45(15): e138, 2017 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-28641402

RESUMO

Purification of cell type-specific RNAs remains a significant challenge. One solution involves biosynthetic tagging of target RNAs. RNA tagging via incorporation of 4-thiouracil (TU) in cells expressing transgenic uracil phosphoribosyltransferase (UPRT), a method known as TU-tagging, has been used in multiple systems but can have limited specificity due to endogenous pathways of TU incorporation. Here, we describe an alternative method that requires the activity of two enzymes: cytosine deaminase (CD) and UPRT. We found that the sequential activity of these enzymes converts 5-ethynylcytosine (EC) to 5-ethynyluridine monophosphate that is subsequently incorporated into nascent RNAs. The ethynyl group allows efficient detection and purification of tagged RNAs. We show that 'EC-tagging' occurs in tissue culture cells and Drosophila engineered to express CD and UPRT. Additional control can be achieved through a split-CD approach in which functional CD is reconstituted from independently expressed fragments. We demonstrate the sensitivity and specificity of EC-tagging by obtaining cell type-specific gene expression data from intact Drosophila larvae, including transcriptome measurements from a small population of central brain neurons. EC-tagging provides several advantages over existing techniques and should be broadly useful for investigating the role of differential RNA expression in cell identity, physiology and pathology.


Assuntos
Linhagem da Célula/genética , Citosina/análogos & derivados , RNA/análise , Coloração e Rotulagem/métodos , Animais , Animais Geneticamente Modificados , Células Cultivadas , Citosina/metabolismo , Citosina/farmacologia , Citosina Desaminase/metabolismo , Drosophila melanogaster , Perfilação da Expressão Gênica/métodos , Células HeLa , Humanos , Especificidade de Órgãos/genética , Pentosiltransferases/metabolismo , RNA/genética
10.
Blood ; 126(14): 1683-94, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26311362

RESUMO

Chromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene occur in primary and treatment-related leukemias and confer a poor prognosis. Studies based primarily on mouse models have substantially advanced our understanding of MLL leukemia pathogenesis, but often use supraphysiological oncogene expression with uncertain implications for human leukemia. Genome editing using site-specific nucleases provides a powerful new technology for gene modification to potentially model human disease, however, this approach has not been used to re-create acute leukemia in human cells of origin comparable to disease observed in patients. We applied transcription activator-like effector nuclease-mediated genome editing to generate endogenous MLL-AF9 and MLL-ENL oncogenes through insertional mutagenesis in primary human hematopoietic stem and progenitor cells (HSPCs) derived from human umbilical cord blood. Engineered HSPCs displayed altered in vitro growth potentials and induced acute leukemias following transplantation in immunocompromised mice at a mean latency of 16 weeks. The leukemias displayed phenotypic and morphologic similarities with patient leukemia blasts including a subset with mixed phenotype, a distinctive feature seen in clinical disease. The leukemic blasts expressed an MLL-associated transcriptional program with elevated levels of crucial MLL target genes, displayed heightened sensitivity to DOT1L inhibition, and demonstrated increased oncogenic potential ex vivo and in secondary transplant assays. Thus, genome editing to create endogenous MLL oncogenes in primary human HSPCs faithfully models acute MLL-rearranged leukemia and provides an experimental platform for prospective studies of leukemia initiation and stem cell biology in a genetic subtype of poor prognosis leukemia.


Assuntos
Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Células-Tronco Hematopoéticas , Histona-Lisina N-Metiltransferase/genética , Leucemia Aguda Bifenotípica/genética , Proteína de Leucina Linfoide-Mieloide/genética , Animais , Antígenos CD34/imunologia , Separação Celular , Técnicas de Introdução de Genes , Genoma Humano , Humanos , Camundongos , Microscopia Confocal , Mutagênese Sítio-Dirigida , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Transdução Genética , Transfecção
11.
Cancer Sci ; 106(3): 227-36, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25529853

RESUMO

Acute myeloid leukemia is a clonal malignant disorder derived from a small number of leukemic stem cells (LSCs). Rearrangements of the mixed lineage leukemia (MLL) gene are found in acute myeloid leukemia associated with poor prognosis. The upregulation of Hox genes is critical for LSC induction and maintenance, but is unlikely to support malignancy and the high LSC frequency observed in MLL leukemias. The present study shows that MLL fusion proteins interact with the transcription factor PU.1 to activate the transcription of CSF-1R, which is critical for LSC activity. Acute myeloid leukemia is cured by either deletion of PU.1 or ablation of cells expressing CSF-1R. Kinase inhibitors specific for CSF-1R prolong survival time. These findings indicate that PU.1-mediated upregulation of CSF-1R is a critical effector of MLL leukemogenesis.


Assuntos
Carcinogênese/genética , Histona-Lisina N-Metiltransferase/genética , Leucemia Mieloide Aguda/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Proto-Oncogênicas/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Transativadores/genética , Animais , Regulação Leucêmica da Expressão Gênica , Genes Homeobox , Leucemia Mieloide Aguda/mortalidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Neoplásicas , Compostos de Fenilureia/farmacologia , Prognóstico , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais , Tacrolimo/análogos & derivados , Tacrolimo/farmacologia , Tiazóis/farmacologia , Transcrição Gênica , Ativação Transcricional , Regulação para Cima
12.
J Cell Sci ; 126(Pt 14): 3181-91, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23660001

RESUMO

The capacity of the hematopoietic system to promptly respond to peripheral demands relies on adequate pools of progenitors able to transiently proliferate and differentiate in a regulated manner. However, little is known about factors that may restrain progenitor maturation to maintain their reservoirs. Conditional knockout mice for the Pbx1 proto-oncogene have a significant reduction in lineage-restricted progenitors in addition to a profound defect in hematopoietic stem cell (HSC) self-renewal. Through analysis of purified progenitor proliferation, differentiation capacity and transcriptional profiling, we demonstrate that Pbx1 regulates the lineage-specific output of multipotent and oligopotent progenitors. In the absence of Pbx1 multipotent progenitor (MPP) and common myeloid progenitor (CMP) pools are reduced due to aberrantly rapid myeloid maturation. This is associated with premature expression of myeloid differentiation genes and decreased maintenance of proto-oncogene transcriptional pathways, including reduced expression of Meis1, a Pbx1 dimerization partner, and its subordinate transcriptional program. Conversely, Pbx1 maintains the lymphoid differentiation potential of lymphoid-primed MPPs (LMPPs) and common lymphoid progenitors (CLPs), whose reduction in the absence of Pbx1 is associated with a defect in lymphoid priming that is also present in CMPs, which persistently express lymphoid and HSC genes underlying a previously unappreciated lineage promiscuity that is maintained by Pbx1. These results demonstrate a role for Pbx1 in restraining myeloid maturation while maintaining lymphoid potential to appropriately regulate progenitor reservoirs.


Assuntos
Hematopoese , Proteínas de Homeodomínio/metabolismo , Células Progenitoras Linfoides/fisiologia , Células Progenitoras Mieloides/fisiologia , Proteínas de Neoplasias/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem da Célula/genética , Sobrevivência Celular/genética , Células Cultivadas , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Proteína Meis1 , Fator de Transcrição 1 de Leucemia de Células Pré-B , Multimerização Proteica , Fatores de Transcrição/genética , Ativação Transcricional
13.
Development ; 139(4): 657-66, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22219354

RESUMO

Similar to mammalian neural progenitors, Drosophila neuroblasts progressively lose competence to make early-born neurons. In neuroblast 7-1 (NB7-1), Kruppel (Kr) specifies the third-born U3 motoneuron and Kr misexpression induces ectopic U3 cells. However, competence to generate U3 cells is limited to early divisions, when the Eve(+) U motoneurons are produced, and competence is lost when NB7-1 transitions to making interneurons. We have found that Polycomb repressor complexes (PRCs) are necessary and sufficient to restrict competence in NB7-1. PRC loss of function extends the ability of Kr to induce U3 fates and PRC gain of function causes precocious loss of competence to make motoneurons. PRCs also restrict competence to make HB9(+) Islet(+) motoneurons in another neuroblast that undergoes a motoneuron-to-interneuron transition, NB3-1. In contrast to the regulation of motoneuron competence, PRC activity does not affect the production of Eve(+) interneurons by NB3-3, HB9(+) Islet(+) interneurons by NB7-3, or Dbx(+) interneurons by multiple neuroblasts. These findings support a model in which PRCs establish motoneuron-specific competence windows in neuroblasts that transition from motoneuron to interneuron production.


Assuntos
Diferenciação Celular/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomia & histologia , Neurônios Motores/fisiologia , Complexos Multiproteicos/metabolismo , Animais , Linhagem da Célula , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Epigênese Genética , Interneurônios/citologia , Interneurônios/fisiologia , Neurônios Motores/citologia , Complexos Multiproteicos/química , Mutação , Complexo Repressor Polycomb 1
14.
Molecules ; 20(7): 12280-99, 2015 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-26154886

RESUMO

Analyses of key odorous polyfunctional volatile thiols in wines (3-mercaptohexanol (3-MH), 3-mercaptohexylacetate (3-MHA), and 4-mercapto-4-methyl-2-pentanone (4-MMP)) are challenging due to their high reactivity and ultra-trace concentrations, especially when using conventional gas-chromatography electron impact mass spectrometry (GC-EI-MS). We describe a method in which thiols are converted to pentafluorobenzyl (PFB) derivatives by extractive alkylation and the organic layer is evaporated prior to headspace solid phase microextraction (HS-SPME) and GC-EI-MS analysis. Optimal parameters were determined by response surface area modeling. The addition of NaCl solution to the dried SPME vials prior to extraction resulted in up to less than fivefold improvement in detection limits. Using 40 mL wine samples, limits of detection for 4-MMP, 3-MH, and 3-MHA were 0.9 ng/L, 1 ng/L, and 17 ng/L, respectively. Good recovery (90%-109%) and precision (5%-11% RSD) were achieved in wine matrices. The new method was used to survey polyfunctional thiol concentrations in 61 commercial California and New York State wines produced from V. vinifera (Riesling, Gewürztraminer, Cabernet Sauvignon, Sauvignon blanc and non-varietal rosé wines), V. labruscana (Niagara), and Vitis spp. (Cayuga White). Mean 4-MMP concentrations in New York Niagara (17 ng/L) were not significantly different from concentrations in Sauvignon blanc, but were significantly higher than 4-MMP in other varietal wines.


Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Microextração em Fase Sólida/métodos , Compostos de Sulfidrila/análise , Vinho/análise , Alquilação , Limite de Detecção , Reprodutibilidade dos Testes
15.
Dev Biol ; 376(2): 150-62, 2013 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-23376107

RESUMO

Neural progenitors of the Drosophila larval brain, called neuroblasts, can be divided into distinct populations based on patterns of proliferation and differentiation. Type I neuroblasts produce ganglion mother cells (GMCs) that divide once to produce differentiated progeny, while type II neuroblasts produce self-renewing intermediate neural progenitors (INPs) and thus generate lineages containing many more progeny. We identified Taranis (Tara) as an important determinant of type I lineage-specific neural progenitor proliferation patterns. Tara is an ortholog of mammalian SERTAD proteins that are known to regulate cell cycle progression. Tara is differentially-expressed in neural progenitors, with high levels of expression in proliferating type I neuroblasts but no detectable expression in type II lineage INPs. Tara is necessary for cell cycle reactivation in quiescent neuroblasts and for cell cycle progression in type I lineages. Cell cycle defects in tara mutant neuroblasts are due to decreased activation of the E2F1/Dp transcription factor complex and delayed progression through S-phase. Mis-expression of tara in type II lineages delays INP cell cycle progression and induces premature differentiation of INPs into GMCs. Premature INP differentiation can also be induced by loss of E2F1/Dp function and elevated E2F1/Dp expression suppresses Tara-induced INP differentiation. Our results show that lineage-specific Tara expression is necessary for proper brain development and suggest that distinct cell cycle regulatory mechanisms exist in type I versus type II neural progenitors.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Neurônios/citologia , Células-Tronco/citologia , Animais , Ciclo Celular , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Cruzamentos Genéticos , Drosophila melanogaster , Perfilação da Expressão Gênica , Imuno-Histoquímica/métodos , Hibridização In Situ , Células-Tronco Neurais/citologia , Neurônios/metabolismo , Fatores de Tempo
16.
Genome Res ; 21(5): 798-810, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21451113

RESUMO

Emerging evidence suggests that microRNAs (miRNAs), an abundant class of ∼22-nucleotide small regulatory RNAs, play key roles in controlling the post-transcriptional genetic programs in stem and progenitor cells. Here we systematically examined miRNA expression profiles in various adult tissue-specific stem cells and their differentiated counterparts. These analyses revealed miRNA programs that are common or unique to blood, muscle, and neural stem cell populations and miRNA signatures that mark the transitions from self-renewing and quiescent stem cells to proliferative and differentiating progenitor cells. Moreover, we identified a stem/progenitor transition miRNA (SPT-miRNA) signature that predicts the effects of genetic perturbations, such as loss of PTEN and the Rb family, AML1-ETO9a expression, and MLL-AF10 transformation, on self-renewal and proliferation potentials of mutant stem/progenitor cells. We showed that some of the SPT-miRNAs control the self-renewal of embryonic stem cells and the reconstitution potential of hematopoietic stem cells (HSCs). Finally, we demonstrated that SPT-miRNAs coordinately regulate genes that are known to play roles in controlling HSC self-renewal, such as Hoxb6 and Hoxa4. Together, these analyses reveal the miRNA programs that may control key processes in normal and aberrant stem and progenitor cells, setting the foundations for dissecting post-transcriptional regulatory networks in stem cells.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica/genética , MicroRNAs/metabolismo , Células-Tronco/metabolismo , Animais , Diferenciação Celular/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , MicroRNAs/genética , Mutação , Mioblastos/citologia , Mioblastos/metabolismo , Células-Tronco Neurais , Especificidade de Órgãos , Células-Tronco/citologia
17.
Nat Cell Biol ; 9(10): 1208-15, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17891136

RESUMO

Enzymes that mediate reversible epigenetic modifications have not only been recognized as key in regulating gene expression and oncogenesis, but also provide potential targets for molecular therapy. Although the methylation of arginine 3 of histone 4 (H4R3) by protein arginine methyltransferase 1 (PRMT1) is a critical modification for active chromatin and prevention of heterochromatin spread, there has been no direct evidence of any role of PRMTs in cancer. Here, we show that PRMT1 is an essential component of a novel Mixed Lineage Leukaemia (MLL) oncogenic transcriptional complex with both histone acetylation and H4R3 methylation activities, which also correlate with the expression of critical MLL downstream targets. Direct fusion of MLL with PRMT1 or Sam68, a bridging molecule in the complex for PRMT1 interaction, could enhance self-renewal of primary haematopoietic cells. Conversely, specific knockdown of PRMT1 or Sam68 expression suppressed MLL-mediated transformation. This study not only functionally dissects the oncogenic transcriptional machinery associated with an MLL fusion complex, but also uncovers--for the first time--an essential function of PRMTs in oncogenesis and reveals their potential as novel therapeutic targets in human cancer.


Assuntos
Transformação Celular Neoplásica/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Acetilação , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Arginina/metabolismo , Linhagem Celular , Transformação Celular Neoplásica/genética , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Imunofluorescência , Regulação da Expressão Gênica , Células HeLa , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Imunoprecipitação , Metilação , Mutação , Regiões Promotoras Genéticas/genética , Proteína-Arginina N-Metiltransferases/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
18.
Cancer Cell ; 10(4): 257-68, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17045204

RESUMO

Using a mouse model of human acute myeloid leukemia (AML) induced by the MLL-AF9 oncogene, we demonstrate that colony-forming cells (CFCs) in the bone marrow and spleen of leukemic mice are also leukemia stem cells (LSCs). These self-renewing cells (1) are frequent, accounting for 25%-30% of myeloid lineage cells at late-stage disease; (2) generate a phenotypic, morphologic, and functional leukemia cell hierarchy; (3) express mature myeloid lineage-specific antigens; and (4) exhibit altered microenvironmental interactions by comparison with the oncogene-immortalized CFCs that initiated the disease. Therefore, the LSCs responsible for sustaining, expanding, and regenerating MLL-AF9 AML are downstream myeloid lineage cells, which have acquired an aberrant Hox-associated self-renewal program as well as other biologic features of hematopoietic stem cells.


Assuntos
Células-Tronco Hematopoéticas/patologia , Leucemia Experimental/patologia , Leucemia Mieloide Aguda/genética , Células Mieloides/patologia , Proteínas de Fusão Oncogênica/metabolismo , Animais , Células da Medula Óssea/citologia , Técnicas de Cultura de Células , Linhagem Celular Transformada , Linhagem da Célula , Transformação Celular Neoplásica , Técnicas de Cocultura , Meios de Cultivo Condicionados , Modelos Animais de Doenças , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunofenotipagem , Leucemia Experimental/etiologia , Leucemia Experimental/genética , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Transgênicos , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Retroviridae/genética , Baço/patologia , Transdução Genética , Transplante Homólogo , Raios X
19.
Cancer Cell ; 10(6): 456-7, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17157786

RESUMO

Improved understanding of the molecular pathways that suppress the genesis and maintenance of cancer stem cells will facilitate development of rationally targeted therapies. PU.1 is a transcription factor that is required for normal myelomonocytic differentiation in hematopoiesis, and reduced PU.1 activity has been associated with myeloid leukemogenesis in man and in mouse models. A recent study by Steidl et al. demonstrates that Junb and Jun, two AP-1 transcription factors, are critical downstream effectors of the tumor suppressor activity of PU.1, and that reduced expression of Junb, in particular, may be a common feature of acute myeloid leukemogenesis.


Assuntos
Leucemia Mieloide Aguda/prevenção & controle , Células-Tronco Neoplásicas/fisiologia , Proteínas Proto-Oncogênicas c-jun/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Transativadores/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Humanos , Camundongos
20.
Nature ; 455(7217): 1205-9, 2008 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-18806775

RESUMO

Glycogen synthase kinase 3 (GSK3) is a multifunctional serine/threonine kinase that participates in numerous signalling pathways involved in diverse physiological processes. Several of these pathways are implicated in disease pathogenesis, which has prompted efforts to develop GSK3-specific inhibitors for therapeutic applications. However, before now, there has been no strong rationale for targeting GSK3 in malignancies. Here we report pharmacological, physiological and genetic studies that demonstrate an oncogenic requirement for GSK3 in the maintenance of a specific subtype of poor prognosis human leukaemia, genetically defined by mutations of the MLL proto-oncogene. In contrast to its previously characterized roles in suppression of neoplasia-associated signalling pathways, GSK3 paradoxically supports MLL leukaemia cell proliferation and transformation by a mechanism that ultimately involves destabilization of the cyclin-dependent kinase inhibitor p27(Kip1). Inhibition of GSK3 in a preclinical murine model of MLL leukaemia provides promising evidence of efficacy and earmarks GSK3 as a candidate cancer drug target.


Assuntos
Transformação Celular Neoplásica , Quinase 3 da Glicogênio Sintase/metabolismo , Leucemia Linfoide/tratamento farmacológico , Leucemia Linfoide/patologia , Proteína de Leucina Linfoide-Mieloide/metabolismo , Animais , Divisão Celular , Linhagem Celular Transformada , Linhagem Celular Tumoral , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27 , Modelos Animais de Doenças , Fase G1 , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/deficiência , Quinase 3 da Glicogênio Sintase/genética , Histona-Lisina N-Metiltransferase , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isoenzimas/metabolismo , Leucemia Linfoide/enzimologia , Leucemia Linfoide/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Células Progenitoras Mieloides/enzimologia , Células Progenitoras Mieloides/metabolismo , Células Progenitoras Mieloides/patologia , Células Precursoras de Linfócitos B/enzimologia , Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/patologia , Proto-Oncogene Mas
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