RESUMO
Hematologic malignancies are often associated with chromosomal rearrangements that lead to the expression of chimeric fusion proteins. Rearrangements of the genes encoding two nucleoporins, NUP98 and NUP214, have been implicated in the pathogenesis of several types of hematologic malignancies, particularly acute myeloid leukemia. NUP98 rearrangements result in fusion of an N-terminal portion of NUP98 to one of numerous proteins. These rearrangements often follow treatment with topoisomerase II inhibitors and tend to occur in younger patients. They have been shown to induce leukemia in mice and to enhance proliferation and disrupt differentiation in primary human hematopoietic precursors. NUP214 has only a few fusion partners. DEK-NUP214 is the most common NUP214 fusion in AML; it tends to occur in younger patients and is usually associated with FLT3 internal tandem duplications. The leukemogenic activity of NUP214 fusions is less well characterized. Normal nucleoporins, including NUP98 and NUP214, have important functions in nucleocytoplasmic transport, transcription, and mitosis. These functions and their disruptions by oncogenic nucleoporin fusions are discussed.
Assuntos
Neoplasias Hematológicas/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Neoplasias Hematológicas/genética , Humanos , Mitose , Transcrição GênicaRESUMO
A substantial body of evidence points to the heritability of dietary preferences. While vegetarianism has been practiced for millennia in various societies, its practitioners remain a small minority of people worldwide, and the role of genetics in choosing a vegetarian diet is not well understood. Dietary choices involve an interplay between the physiologic effects of dietary items, their metabolism, and taste perception, all of which are strongly influenced by genetics. In this study, we used a genome-wide association study (GWAS) to identify loci associated with strict vegetarianism in UK Biobank participants. Comparing 5,324 strict vegetarians to 329,455 controls, we identified one SNP on chromosome 18 that is associated with vegetarianism at the genome-wide significant level (rs72884519, ß = -0.11, P = 4.997 x 10-8), and an additional 201 suggestively significant variants. Four genes are associated with rs72884519: TMEM241, RIOK3, NPC1, and RMC1. Using the Functional Mapping and Annotation (FUMA) platform and the Multi-marker Analysis of GenoMic Annotation (MAGMA) tool, we identified 34 genes with a possible role in vegetarianism, 3 of which are GWAS-significant based on gene-level analysis: RIOK3, RMC1, and NPC1. Several of the genes associated with vegetarianism, including TMEM241, NPC1, and RMC1, have important functions in lipid metabolism and brain function, raising the possibility that differences in lipid metabolism and their effects on the brain may underlie the ability to subsist on a vegetarian diet. These results support a role for genetics in choosing a vegetarian diet and open the door to future studies aimed at further elucidating the physiologic pathways involved in vegetarianism.
Assuntos
Dieta Vegetariana , Estudo de Associação Genômica Ampla , Humanos , Dieta , Dieta Vegana , EncéfaloRESUMO
NUP98-HOXA9 is the prototype of NUP98 fusion oncoproteins that cause acute myeloid leukemia. It consists of an N-terminal FG-rich portion of the nucleoporin NUP98 fused to the homeodomain region of the homeobox protein HOXA9, and acts as an aberrant transcription factor. To identify interacting partners of NUP98-HOXA9, we used a cytoplasmic yeast two-hybrid assay to avoid the nonspecific trans-activation that would occur with the traditional yeast two-hybrid assay due to the transactivating properties of NUP98-HOXA9. We identified amino-terminal enhancer of split (AES), a transcriptional regulator of the transducin-like enhancer/Groucho family as a novel interaction partner of NUP98-HOXA9. The interaction was confirmed by in vitro pulldown and co-immunoprecipitation assays and was shown to require the FG repeat region of NUP98-HOXA9. Immunofluorescence analysis showed that AES localizes primarily to the interior of the nucleus. AES also showed a strong interaction with wild-type NUP98. AES augmented the transcriptional activity of NUP98-HOXA9. In the presence of NUP98-HOXA9, AES caused an increase in long-term proliferation of primary human CD34+ cells with a marked increase in the numbers of primitive cells. These effects of AES were not observed in the absence of NUP98-HOXA9. AES knockdown diminished the transcriptional and proliferative effects of NUP98-HOXA9. AES caused a shift away from the erythroid lineage in cells expressing NUP98-HOXA9. These data establish AES as an interacting partner of NUP98-HOXA9 and show that it cooperates with NUP98-HOXA9 in transcriptional regulation and cell transformation.
Assuntos
Núcleo Celular/metabolismo , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Proteínas de Homeodomínio/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Antígenos CD34 , Núcleo Celular/genética , Núcleo Celular/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Proteínas Correpressoras , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Humanos , Células K562 , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Ligação Proteica , Proteínas Repressoras/genética , Saccharomyces cerevisiae , Técnicas do Sistema de Duplo-HíbridoRESUMO
OBJECTIVES: Acute myeloid leukemia (AML) with t(8;16)(p11;p13) abnormalities is a rare, aggressive, and diagnostically challenging subtype that results in KAT6A-CREBBP gene fusion. METHODS: To investigate their immunophenotype and genomic features, we identified 5 cases of AML with t(8;16) through a retrospective review of the databases at Northwestern Memorial Hospital in Chicago, IL, and Washington University Medical Center, in St Louis, MO. RESULTS: In all, 4 of 5 cases were therapy related and 1 was possibly therapy related. The leukemic blasts showed distinctive features, including bright CD45 expression and remarkably high side scatter that overlapped with maturing myeloid elements, making the blasts difficult to identify on initial examination. They were positive for CD13, CD33, and CD64 and negative for CD34 and CD117. Next-generation sequencing profiling of 4 cases revealed pathogenic ASXL1 (2 cases), FLT3-tyrosine kinase domain (TKD) mutations (2 cases), and other pathogenic mutations. In 3 patients, t(8;16) was the sole cytogenetic abnormality; additional aberrations were found in 2 patients. Single nucleotide polymorphism microarray revealed 1 case with 7q deletion as a secondary clone. CONCLUSIONS: Our data highlight the distinctive immunophenotypic profile of AML with t(8;16), which, along with its unique morphology, often presents a diagnostic challenge. We showed that mutations of either ASXL1 or FLT3-TKD are seen in most cases of this leukemia.
Assuntos
Leucemia Mieloide Aguda , Aberrações Cromossômicas , Citometria de Fluxo/métodos , Humanos , Imunofenotipagem , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Mutação , Translocação GenéticaRESUMO
NUP98 is a nucleoporin that plays complex roles in the nucleocytoplasmic trafficking of macromolecules. Rearrangements of the NUP98 gene in human leukemia result in the expression of numerous fusion oncoproteins whose effect on nucleocytoplasmic trafficking is poorly understood. The present study was undertaken to determine the effects of leukemogenic NUP98 fusion proteins on CRM1-mediated nuclear export. NUP98-HOXA9, a prototypic NUP98 fusion, inhibited the nuclear export of two known CRM1 substrates: mutated cytoplasmic nucleophosmin and HIV-1 Rev. In vitro binding assays revealed that NUP98-HOXA9 binds CRM1 through the FG repeat motif in a Ran-GTP-dependent manner similar to but stronger than the interaction between CRM1 and its export substrates. Two NUP98 fusions, NUP98-HOXA9 and NUP98-DDX10, whose fusion partners are structurally and functionally unrelated, interacted with endogenous CRM1 in myeloid cells as shown by co-immunoprecipitation. These leukemogenic NUP98 fusion proteins interacted with CRM1, Ran, and the nucleoporin NUP214 in a manner fundamentally different from that of wild-type NUP98. NUP98-HOXA9 and NUP98-DDX10 formed characteristic aggregates within the nuclei of a myeloid cell line and primary human CD34+ cells and caused aberrant localization of CRM1 to these aggregates. These NUP98 fusions caused nuclear accumulation of two transcription factors, NFAT and NFkappaB, that are regulated by CRM1-mediated export. The nuclear entrapment of NFAT and NFkappaB correlated with enhanced transcription from promoters responsive to these transcription factors. Taken together, the results suggest a new mechanism by which NUP98 fusions dysregulate transcription and cause leukemia, namely, inhibition of CRM1-mediated nuclear export with aberrant nuclear retention of transcriptional regulators.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Homeodomínio/metabolismo , Carioferinas/metabolismo , Leucemia/metabolismo , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transporte Ativo do Núcleo Celular/genética , Motivos de Aminoácidos , Antígenos CD34 , Núcleo Celular/genética , Núcleo Celular/patologia , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , HIV-1/genética , HIV-1/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Células K562 , Carioferinas/genética , Leucemia/genética , Leucemia/patologia , Mutação , NF-kappa B/genética , Fatores de Transcrição NFATC/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Regiões Promotoras Genéticas/genética , Receptores Citoplasmáticos e Nucleares/genética , Transcrição Gênica/genética , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismo , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Proteína Exportina 1RESUMO
EBV-negative aggressive NK-cell leukemia/lymphoma (ANKL) is a recently recognized, rare NK-cell neoplasm that preferentially affects non-Asians and has a fulminant clinical course. Little is known about the genetic alterations of this disease. In this study, we performed comprehensive molecular genetic studies, including chromosomal analysis, fluorescence in situ hybridization, single nucleotide polymorphism (SNP) microarray, and next-generation sequencing (NGS), on 4 patients diagnosed in our institution. The results demonstrated that our EBV-negative ANKLs have highly complex genomic profiles characterized by near-triploid/near-tetraploid karyotype (3 of 3) with numerous structural abnormalities, inactivation of TP53 (3 of 3), overexpression of c-Myc (4 of 4), strong expression of PD-L1 in neoplastic cells (2 of 4), and gain of the 11q23-ter region (2 of 2). Our study provides important insights of EBV-negative ANKL, which share many of the genetic features with their EBV-positive counterpart. The strong expression of Programmed death-ligand 1 (PD-L1) suggests that immune checkpoint inhibitors may be further explored as a potential therapeutic option for this highly aggressive, chemotherapy-resistant NK-cell neoplasm.
Assuntos
Biomarcadores Tumorais/genética , Leucemia Linfocítica Granular Grande/genética , Técnicas de Diagnóstico Molecular , Idoso , Idoso de 80 Anos ou mais , Antígeno B7-H1/genética , Feminino , Amplificação de Genes , Inativação Gênica , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Leucemia Linfocítica Granular Grande/patologia , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-myc/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Establishing the pathologic diagnosis of central nervous system (CNS) lymphoma can be challenging, yet management of this potentially curable disease depends heavily on it. One avoidable impediment to obtaining an accurate and timely diagnosis is the pre-operative administration of steroids, which causes tumor involution and prevents appropriate sampling of viable tissue. We discuss a case of primary CNS lymphoma that highlights the evolution of the disease and the attempts to establish a diagnosis in the setting of prior administration of corticosteroids. Familiarity with these clinical scenarios will help others avoid delays in patient care that results from delayed diagnosis.
Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Dexametasona/administração & dosagem , Glucocorticoides/administração & dosagem , Linfoma Difuso de Grandes Células B/diagnóstico por imagem , Idoso , Neoplasias Encefálicas/sangue , Neoplasias Encefálicas/tratamento farmacológico , Humanos , Linfoma Difuso de Grandes Células B/sangue , Linfoma Difuso de Grandes Células B/tratamento farmacológico , MasculinoRESUMO
CONTEXT.: The Accreditation Council for Graduate Medical Education (ACGME) established a new system for accreditation of residency and fellowship programs in 2013. One key aspect of the Next Accreditation System is the 10-year self-study, which requires programs to conduct a comprehensive self-evaluation, including development of program aims and analysis of strengths, weaknesses, and environmental context, in order to plan improvements and take the program to the next level. OBJECTIVE.: To provide a review of the recent changes and current state of ACGME accreditation, with a focus on the new 10-year self-study, and to share our institution's experience with conducting the first self-study of our pathology residency and accredited fellowship programs in 2018. DATA SOURCES.: Review of English-language literature, published resources from the ACGME, and materials/data from our department's 2018 self-study. CONCLUSIONS.: The self-study process now required for ACGME accreditation is a useful way to assess program strengths and weaknesses in the context of current environmental and institutional factors, and helps develop an effective framework for improvements geared at achieving program aims and taking the program to the next level. Additionally, conducting residency and fellowship self-studies together allows for collaboration, effective use of shared resources, and the development of a cohesive educational mission.
Assuntos
Acreditação , Educação de Pós-Graduação em Medicina/normas , Patologia/educação , Bolsas de Estudo , Humanos , Internato e ResidênciaRESUMO
NUP98-HOXA9, the chimeric protein resulting from the t(7;11)(p15;p15) chromosomal translocation, is a prototype of several NUP98 fusions that occur in myelodysplastic syndromes and acute myeloid leukemia. We examined its effect on differentiation, proliferation, and gene expression in primary human CD34+ hematopoietic cells. Colony-forming cell (CFC) assays in semisolid medium combined with morphologic examination and flow cytometric immunophenotyping revealed that NUP98-HOXA9 increased the numbers of erythroid precursors and impaired both myeloid and erythroid differentiation. In continuous liquid culture, cells transduced with NUP98-HOXA9 exhibited a biphasic growth curve with initial growth inhibition followed by enhanced long-term proliferation, suggesting an increase in the numbers of primitive self-renewing cells. This was confirmed by a dramatic increase in the numbers of long-term culture-initiating cells, the most primitive hematopoietic cells detectable in vitro. To understand the molecular mechanisms underlying the effects of NUP98-HOXA9 on hematopoietic cell proliferation and differentiation, oligonucleotide microarray analysis was done at several time points over 16 days, starting at 6 hours posttransduction. The early growth suppression was preceded by up-regulation of IFNbeta1 and accompanied by marked up-regulation of IFN-induced genes, peaking at 3 days posttransduction. In contrast, oncogenes such as homeobox transcription factors, FLT3, KIT, and WT1 peaked at 8 days or beyond, coinciding with increased proliferation. In addition, several putative tumor suppressors and genes associated with hematopoietic differentiation were repressed at later time points. These findings provide a comprehensive picture of the changes in proliferation, differentiation, and global gene expression that underlie the leukemic transformation of human hematopoietic cells by NUP98-HOXA9.
Assuntos
Antígenos CD34/biossíntese , Células-Tronco Hematopoéticas/fisiologia , Proteínas de Homeodomínio/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Proteínas de Fusão Oncogênica/fisiologia , Diferenciação Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Células Eritroides/citologia , Células Eritroides/metabolismo , Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Interferon beta/genética , Células Mieloides/citologia , Células Mieloides/metabolismo , Células Mieloides/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/biossíntese , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/biossíntese , Proteínas de Fusão Oncogênica/genética , Retroviridae/genética , Fatores de Transcrição/genética , Transdução GenéticaRESUMO
A 29-year-old woman presented with blurred vision and distal paresthesias. Her initial evaluation revealed severe bilateral optic disc edema with distal lower-extremity sensory and motor deficits and electrodiagnostic evidence of a length-dependent mixed demyelinating and axonal polyneuropathy. The results of routine diagnostic testing, including laboratory tests, magnetic resonance imaging, and lumbar puncture, were nondiagnostic. A targeted biopsy was ultimately required for diagnosis. In this article, we discuss the differential diagnosis and outline the clinical evaluation indicated for a patient presenting with demyelinating polyneuropathy and concurrent papilledema.
Assuntos
Parestesia/complicações , Parestesia/diagnóstico , Transtornos da Visão/complicações , Transtornos da Visão/diagnóstico , Adulto , Diagnóstico Diferencial , Feminino , Humanos , Angiografia por Ressonância Magnética , Imageamento por Ressonância Magnética , Tomógrafos Computadorizados , Fator A de Crescimento do Endotélio Vascular/sangueRESUMO
The chimeric oncoprotein NUP98-HOXA9 results from the t(7;11)(p15;p15) chromosomal translocation and is associated with acute myeloid leukemia. It causes aberrant gene regulation and leukemic transformation through mechanisms that are not fully understood. NUP98-HOXA9 consists of an N-terminal portion of the nucleoporin NUP98 that contains many FG repeats fused to the DNA-binding homeodomain of HOXA9. We used a Cytotrap yeast two-hybrid assay to identify proteins that interact with NUP98-HOXA9. We identified Dynein Light Chain 1 (DYNLT1), an integral 14 KDa protein subunit of the large microtubule-based cytoplasmic dynein complex, as an interaction partner of NUP98-HOXA9. Binding was confirmed by in vitro pull down and co-immunoprecipitation assays and the FG repeat region of NUP98-HOXA9 was shown to be essential for the interaction. RNAi-mediated knockdown of DYNLT1 resulted in reduction of the ability of NUP98-HOXA9 to activate transcription and also inhibited the ability of NUP98-HOXA9 to induce proliferation of primary human hematopoietic CD34+ cells. DYNLT1 also showed a strong interaction with wild-type NUP98 and other nucleoporins containing FG repeats. Immunofluorescence analysis showed that DYNLT1 localizes primarily to the nuclear periphery, where it co-localizes with the nuclear pore complex, and to the cytoplasm. Deletion studies showed that the interactions of the nucleoporins with DYNLT1 are dependent predominantly on the C-terminal half of the DYNLT1. These data show for the first time that DYNLT1 interacts with nucleoporins and plays a role in the dysregulation of gene expression and induction of hematopoietic cell proliferation by the leukemogenic nucleoporin fusion, NUP98-HOXA9.
Assuntos
Dineínas/metabolismo , Proteínas de Homeodomínio/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Dineínas/deficiência , Dineínas/genética , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Humanos , Células K562 , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Ligação Proteica , Transporte Proteico , Sequências Repetitivas de Aminoácidos , Ativação TranscricionalRESUMO
Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages. We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.
Assuntos
Ensaio de Unidades Formadoras de Colônias/métodos , Células-Tronco Hematopoéticas/citologia , Antígenos CD34/biossíntese , Diferenciação Celular/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Oncogenes , Transdução Genética/métodosRESUMO
Different fusion oncogenes in acute myeloid leukemia (AML) have distinct clinical and laboratory features suggesting different modes of malignant transformation. Here we compare the in vitro effects of representatives of 4 major groups of AML fusion oncogenes on primary human CD34+ cells. As expected from their clinical similarities, MLL-AF9 and NUP98-HOXA9 had very similar effects in vitro. They both caused erythroid hyperplasia and a clear block in erythroid and myeloid maturation. On the other hand, AML1-ETO and PML-RARA had only modest effects on myeloid and erythroid differentiation. All oncogenes except PML-RARA caused a dramatic increase in long-term proliferation and self-renewal. Gene expression profiling revealed two distinct temporal patterns of gene deregulation. Gene deregulation by MLL-AF9 and NUP98-HOXA9 peaked 3 days after transduction. In contrast, the vast majority of gene deregulation by AML1-ETO and PML-RARA occurred within 6 hours, followed by a dramatic drop in the numbers of deregulated genes. Interestingly, the p53 inhibitor MDM2 was upregulated by AML1-ETO at 6 hours. Nutlin-3, an inhibitor of the interaction between MDM2 and p53, specifically inhibited the proliferation and self-renewal of primary human CD34+ cells transduced with AML1-ETO, suggesting that MDM2 upregulation plays a role in cell transformation by AML1-ETO. These data show that differences among AML fusion oncogenes can be recapitulated in vitro using primary human CD34+ cells and that early gene expression profiling in these cells can reveal potential drug targets in AML.
Assuntos
Antígenos CD34/metabolismo , Transformação Celular Neoplásica/genética , Perfilação da Expressão Gênica , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Fusão Oncogênica , Oncogenes/genética , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Fatores de TempoRESUMO
NUP98-HOXA9 is the prototype of a group of oncoproteins associated with acute myeloid leukemia. It consists of an N-terminal portion of NUP98 fused to the homeodomain of HOXA9 and is believed to act as an aberrant transcription factor that binds DNA through the homeodomain. Here we show that NUP98-HOXA9 can regulate transcription without binding to DNA. In order to determine the relative contributions of the NUP98 and HOXA9 portions to the transforming ability of NUP98-HOXA9, the effects of NUP98-HOXA9 on primary human CD34+ cells were dissected and compared to those of wild-type HOXA9. In contrast to previous findings in mouse cells, HOXA9 had only mild effects on the differentiation and proliferation of primary human hematopoietic cells. The ability of NUP98-HOXA9 to disrupt the differentiation of primary human CD34+ cells was found to depend primarily on the NUP98 portion, whereas induction of long-term proliferation required both the NUP98 moiety and an intact homeodomain. Using oligonucleotide microarrays in primary human CD34+ cells, a group of genes was identified whose dysregulation by NUP98-HOXA9 is attributable primarily to the NUP98 portion. These include RAP1A, HEY1, and PTGS2 (COX-2). Their functions may reflect the contribution of the NUP98 moiety of NUP98-HOXA9 to leukemic transformation. Taken together, these results suggest that the effects of NUP98-HOXA9 on gene transcription and cell transformation are mediated by at least two distinct mechanisms: one that involves promoter binding through the homeodomain with direct transcriptional activation, and another that depends predominantly on the NUP98 moiety and does not involve direct DNA binding.
Assuntos
Células da Medula Óssea/citologia , Transformação Celular Neoplásica , Proteínas de Homeodomínio/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Oncogenes , Sequência de Aminoácidos , Sequência de Bases , Imunoprecipitação da Cromatina , Primers do DNA , Citometria de Fluxo , Regulação da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Humanos , Células K562 , Dados de Sequência Molecular , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Transcrição Gênica/fisiologiaRESUMO
Electron microscopy (EM) has been used for several decades to study the mechanisms of nuclear transport. In early studies of nuclear import, gold-conjugated nuclear proteins were microinjected into cells and followed by EM. As the components of the nuclear pore complex (NPC) and soluble mediators of nuclear import were cloned and characterized, gold-conjugated antibodies were utilized to sublocalize the components of the nuclear transport machinery by immuno-EM. Further, gold-conjugated recombinant proteins were used to probe permeabilized cells or isolated nuclear envelopes and characterize binding sites for these proteins at the NPC. More recently, recombinant gold-conjugated nuclear proteins were used in in vitro nuclear import assays to help dissect the mechanisms of nuclear import. We have used this ultrastructural nuclear import assay to study the nuclear import of the transcription factor PU.1. The results showed that this import requires energy but is carrier-independent. In the presence of energy, gold-conjugated PU.1 shifted to the nuclear side of the NPC and the inside of the nucleus. In conjunction with biochemical assays, these results indicated that this shift involved Ran-dependent binding of PU.1 to NUP153, a nucleoporin situated at the nuclear side of the NPC. Here we describe in detail the methods used in the ultrastructural nuclear import assay including preparation of recombinant protein, gold conjugation, in vitro nuclear import assay, electron microscopy, and data analysis.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Bioensaio , Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Núcleo Celular/ultraestrutura , Células HeLa , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , Proteínas Nucleares/ultraestrutura , Transporte Proteico/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/ultraestrutura , Transativadores/metabolismo , Transativadores/ultraestruturaRESUMO
PU.1 is a transcription factor of the Ets family with important functions in hematopoietic cell differentiation. Using green fluorescent protein-PU.1 fusions, we show that the Ets DNA binding domain of PU.1 is necessary and sufficient for its nuclear localization. Fluorescence and ultrastructural nuclear import assays showed that PU.1 nuclear import requires energy but not soluble carriers. PU.1 interacted directly with two nucleoporins, Nup62 and Nup153. The binding of PU.1 to Nup153, but not to Nup62, increased dramatically in the presence of RanGMPPNP, indicating the formation of a PU.1.RanGTP.Nup153 complex. The Ets domain accounted for the bulk of the interaction of PU.1 with Nup153 and RanGMPPNP. Because Nup62 is located close to the midplane of the nuclear pore complex whereas Nup153 is at its nuclear side, these findings suggest a model whereby RanGTP propels PU.1 toward the nuclear side of the nuclear pore complex by increasing its affinity for Nup153. This notion was confirmed by ultrastructural studies using gold-labeled PU.1 in permeabilized cells.
Assuntos
Transporte Ativo do Núcleo Celular , Complexo de Proteínas Formadoras de Poros Nucleares/química , Proteínas Proto-Oncogênicas/química , Transativadores/química , Proteína ran de Ligação ao GTP/química , Transporte Biológico , Núcleo Celular/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Imuno-Histoquímica , Microscopia Eletrônica , Microscopia de Fluorescência , Plasmídeos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
Ran is a small GTPase that cycles between a guanosine diphosphate (GDP)-bound form (RanGDP) and a guanosine triphosphate (GTP)-bound form (RanGTP) and plays important roles in nuclear transport and mitosis. For studies of Ran function and its interactions with partner proteins, pure RanGDP and RanGTP complexes are critical. Ran complexed with the nonhydrolyzable GTP analog, GMPPNP (RanGMPPNP), is used instead of RanGTP when inhibition of hydrolysis is required. In this study, we demonstrate that the binding of Ran to a UNO Q ion exchange column is remarkably sensitive to small shifts in MgCl(2) concentration, and we use this property to purify recombinant RanGTP, RanGMPPNP, and RanGDP complexes. At 10 mM MgCl(2), Ran was found predominantly in the flow-through and, thus, was separated from the vast majority of bacterial proteins. After reducing the concentration of MgCl(2) to 5 mM, further purification of RanGTP, RanGMPPNP, and RanGDP was achieved by loading onto ion exchange columns and elution with an NaCl gradient. Purity of the resulting preparations was confirmed by releasing the bound nucleotide and checking it against a known nucleotide by high-performance liquid chromatography (HPLC). To further confirm the purity and function of the Ran preparations, appropriate protein-binding, enzymatic, and nuclear import assays were carried out. These methods should facilitate studies of cellular processes involving Ran by providing pure functional Ran-nucleotide complexes.
Assuntos
Cromatografia por Troca Iônica/métodos , Nucleotídeos de Guanina/química , Proteína ran de Ligação ao GTP/química , Proteína ran de Ligação ao GTP/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Nucleotídeos de Guanina/metabolismo , Humanos , Cloreto de Magnésio/química , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismoRESUMO
The nucleoporin Nup98 gene is frequently rearranged in acute myelogenous leukemia (AML). In most cases this results in fusion of the N terminus of Nup98 to the DNA binding domain of a homeodomain transcription factor. The prototype of these fusions, Nup98-HOXA9, is associated with human AML and induces AML in mouse models. To understand the mechanisms by which Nup98-HOXA9 causes AML, we expressed it in myeloid cells and identified its target genes using high density oligonucleotide microarrays. The analysis was performed in triplicate and was confirmed by quantitative real time PCR. Of the 102 Nup98-HOXA9 target genes identified, 92 were up-regulated, and only 10 were down-regulated, suggesting a transcriptional activation function. A similar analysis of wild-type HOXA9 revealed 13 target genes, 12 of which were up-regulated, and 1 was down-regulated. In contrast, wild-type Nup98 had no effect on gene expression, demonstrating that the HOXA9 DNA binding domain is required for gene regulation. Co-transfection experiments using a luciferase reporter linked to the promoter of one of the Nup98-HOXA9 target genes confirmed up-regulation at the transcriptional level by Nup98-HOXA9 but not by either HOXA9 or Nup98. These data indicate that Nup98-HOXA9 is an aberrant transcription factor whose activity depends on the HOXA9 DNA binding domain but has a stronger and wider transcriptional effect than HOXA9. Several of the genes regulated by Nup98-HOXA9 are associated with increased cell proliferation and survival as well as drug metabolism, providing insights into the pathogenesis and epidemiology of Nup98-HOXA9-induced AML.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/fisiologia , Células Mieloides/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Proteínas de Fusão Oncogênica/fisiologia , Transcrição Gênica , Animais , Western Blotting , Ciclo Celular , Divisão Celular , Sobrevivência Celular , Regulação para Baixo , Citometria de Fluxo , Genes Reporter , Proteínas de Fluorescência Verde , Humanos , Processamento de Imagem Assistida por Computador , Células K562 , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Luciferases/metabolismo , Proteínas Luminescentes/metabolismo , Camundongos , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional , Transfecção , Regulação para CimaRESUMO
CD45, a receptor-like protein tyrosine phosphatase (PTP), plays an essential role in lymphocyte development and immune responses. Recent evidence suggests that dimerization of CD45 down-regulates its function. However, the mechanisms by which CD45 dimerization is regulated remain unclear, and there is no direct evidence that the PTP activity of CD45 dimers is less than that of monomers. CD45 in lymphocytes associates with CD45-AP (CD45-associated protein). Here we show that T cells from CD45-AP-null mice have a much higher level of CD45 dimers than those of wild-type mice, suggesting that CD45-AP inhibits CD45 dimer formation. This was confirmed with the use of a novel CD45-AP-null T-cell line, ALST-1, that we established from a spontaneous thymic tumor found in a CD45-AP-null mouse. Transfected CD45-AP inhibited CD45 dimer formation in ALST-1 cells in proportion to the amount of CD45-AP expressed. Finally, with the use of microsomal fractions from both mouse thymocytes and ALST-1 transfectants, the PTP activity of CD45 was found to be significantly lower in CD45-AP-negative cells than in CD45-AP-positive cells. Therefore, our results support a model in which binding of CD45-AP to inactive CD45 dimers converts them to active monomers.
Assuntos
Antígenos Comuns de Leucócito/metabolismo , Proteínas de Membrana/fisiologia , Fosfoproteínas/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Animais , Linhagem Celular Tumoral , Dimerização , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Linfócitos T/química , Timo/citologia , Transfecção , Regulação para CimaRESUMO
Acute leukemias demonstrating immunophenotypic features of more than 1 cell lineage are referred to as acute leukemias of ambiguous lineage in the new World Health Organization classification system. A subtype of leukemia of ambiguous lineage is biphenotypic acute leukemia in which the malignant cell population expresses markers of 2 different lineages, most commonly myeloid and either B- or T-lymphoid lineages. This entity has been defined by a scoring system proposed by the European Group for the Immunological Characterization of Acute Leukemias (EGIL), with various markers assigned a score of 2, 1, or 0.5 depending on their specificity for myeloid or lymphoid lineage. Those cases having a score greater than 2 for the myeloid and either the B- or T-lymphoid lineages are biphenotypic acute leukemia in this system. One marker, CD79a, has been so clearly associated with acute lymphoblastic leukemia (ALL) by some researchers that its expression in the presence of blast markers is considered indicative of B-ALL. We describe an unusual case of acute leukemia meeting the criteria for biphenotypic acute leukemia in which CD79a expression was observed in the blast population.