RESUMO
OBJECTIVE: To detect the relative expression of IGLL1 (immunoglobulin lambda-like polypeptide 1) mRNA in bone marrow of children with T-cell acute lymphoblastic leukemia (T-ALL), and analyze its correlation with the clinical characteristics and prognosis of the patients, so as to clarify the clinical significance of IGLL1 in pediatric T-ALL patients. METHODS: A total of 56 pediatric T-ALL patients hospitalized in Children's Hospital of Soochow University from June 2012 to December 2017 and treated with CCLG-ALL 2008 regimen were selected. Transcriptome sequencing technology was used to detect the transcription level of IGLL1 gene in children with T-ALL. According to 25% of the IGLL1 transcription level (cutoff value:448), the enrolled children were divided into IGLL1 low expression group (17 cases) and IGLL1 high expression group (39 cases). Combined with clinical data, the correlation between the expression level of IGLL1 and prognosis of the patients was analyzed. RESULTS: The comparative analysis showed that the transcription level of IGLL1 was not correlated with the clinical characteristics of the patients, such as sex, age, bone marrow blast, white blood cell (WBC) count at initial diagnosis. The 5-year OS rate of patients with high IGLL1 expression was significantly higher than that of patients with low IGLL1 expression (76.9%±6.7% vs 47.1%±12.1%, P =0.018). Further comparison of relapse-free survival (RFS) rate between the two groups showed that the 5-year RFS rate of patients with high IGLL1 expression was higher than that of patients with low IGLL1 expression, but the difference between the two groups was not statistically significant (P =0.095). Multivariate COX analysis was conducted on common clinical prognostic factors (age, sex, WBC count at diagnosis, prednisone response on the 7th day, bone marrow response on the 15th day after treatment) and IGLL1 expression level, and the results showed that IGLL1 expression (P =0.012) and prednisone response (P =0.017) were independent risk factors for overall survival in pediatric T-ALL patients. CONCLUSION: In pediatric T-ALL, the OS rate of children with high expression of IGLL1 gene was significantly higher than that of children with low expression of IGLL1 gene, and the expression level of IGLL1 gene was an independent factor affecting the survival of children with T-ALL, which suggests that IGLL1 is a marker of good clinical prognosis of children with T-ALL.
Assuntos
Cadeias Leves Substitutas da Imunoglobulina , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Criança , Humanos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Relevância Clínica , Intervalo Livre de Doença , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Prednisona/uso terapêutico , Prognóstico , Recidiva , Cadeias Leves Substitutas da Imunoglobulina/genéticaRESUMO
During bone marrow B-cell development, the pre-B-cell receptor is formed by the association of the immunoglobulin heavy chain with a surrogate light chain, which is encoded by the VPREB1, and λ5 genes. It is known that pre-BCR signaling signifies a critical checkpoint at the pre-B-cell stage. Thus, failure pre-BCR signaling is proposed as a critical factor for the development of B-cell acute lymphoblastic leukemia (B-ALL). BALL is the most common pediatric cancer and is one of the leading causes of death in children. Until now, several molecular analyses were performed for genomic alterations in B-ALL, but for genomic analysis of the VPREB1 gene and its rare variations, limited studies have been conducted. In this study, using polymerase chain reaction and direct sequencing of 88 pediatric patients with B-ALL, we investigated the genomic region of the VPREB1 gene to find sequence variations of this gene. Our study presented ten homozygous and heterozygous point mutations and heterozygous nucleotide deletions, in the VPREB1 gene in 36 boys and 32 girls' patients. Our Bioinformatics assay results presented that these variations may alter the RNA folding, protein structure, and therefore probable effect on the protein function. These results propose that nucleotide changes probably contribute to B-ALL pathogenesis.
Assuntos
Linfoma de Burkitt , Leucemia-Linfoma Linfoblástico de Células Precursoras , Linfoma de Burkitt/genética , Criança , Feminino , Humanos , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves Substitutas da Imunoglobulina/genética , Masculino , Glicoproteínas de Membrana , Nucleotídeos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMO
Immunotherapies directed against B-cell surface markers have been a common developmental strategy to treat B-cell malignancies. The immunoglobulin heavy chain surrogate light chain (SLC), comprising the VpreB1 (CD179a) and Lamda5 (CD179b) subunits, is expressed on pro- and pre-B cells, where it governs pre-B-cell receptor (BCR)-mediated autonomous survival signaling. We hypothesized that the pre-BCR might merit the development of targeted immunotherapies to decouple "autonomous" signaling in B-lineage acute lymphoblastic leukemia (B-ALL). We used the Children's Oncology Group (COG) minimal residual disease (MRD) flow panel to assess pre-BCR expression in 36 primary patient samples accrued to COG standard- and high-risk B-ALL studies through AALL03B1. We also assessed CD179a expression in 16 cases with day 29 end-induction samples, preselected to have ≥1% MRD. All analyses were performed on a 6-color Becton-Dickinson flow cytometer in a Clinical Laboratory Improvement Amendment/College of American Pathologist-certified laboratory. Among 36 cases tested, 32 cases were at the pre-B and 4 cases were at the pro-B stages of developmental arrest. One or both monoclonal antibodies (mAbs) showed that CD179a was present in ≥20% of the B-lymphoblast population. All cases expressed CD179a in the end-induction B-lymphoblast population. The CD179a component of the SLC is commonly expressed in B-ALL, regardless of genotype, stage of developmental arrest, or National Cancer Institute risk status.
Assuntos
Linfoma de Burkitt , Linfoma de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Linfócitos B , Linfoma de Burkitt/patologia , Criança , Humanos , Cadeias Leves Substitutas da Imunoglobulina/genética , Cadeias Leves Substitutas da Imunoglobulina/metabolismo , Linfoma de Células B/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Células Precursoras de Linfócitos BRESUMO
BACKGROUND: Multiple Myeloma (MM) is a heterogeneous, hematological neoplasm that accounts 2% of all cancers. Although, autologous stem cell transplantation and chemotherapy are currently the most effective therapy, it carries a notable hazards, in addition for being non curative. Recently, the Clustered Regular Interspaced Short Palindromic Repeats (CRISPR-cas9) has been successfully tried at the experimental level, for the treatment of several hematological malignancies. OBJECTIVES: We aimed to investigate the in-vitro effect of CRISPR-cas9-mediated knock-out of V-set pre B-cell surrogate light chain 1"VPREB1" gene on the malignant proliferation of primary cultured myeloma cells. METHODS: Bioinformatics' analysis was performed to explore the gene expression profile of MM, and the VPREB1 gene was selected as a target gene for this study. We knocked-out the VPREB1 gene in primary cultured myeloma cells using CRISPR-cas9, the VPREB1 gene editing efficacy was verified by determining VPREB1 gene expression at both the mRNA and protein levels by qPCR and immunofluorescence, respectively. Furthermore, the cytotoxic effect on primary myeloma cells proliferation was evaluated using cytotoxicity assay. RESULTS: There was a statistically significant reduction of both VPREB1 mRNA and protein expression levels (p<0.01). knock-out of VPREB1 gene in myeloma cell line resulted in a statistically significant reduction of myeloma cell proliferation. CONCLUSION: CRISPR-cas9-mediated knock-out of VPREB1 gene is effective for inhibiting the proliferation of primary myeloma cells. This would provide a basis for a promising therapeutic strategy for patients with multiple myeloma.
Assuntos
Sistemas CRISPR-Cas , Cadeias Leves Substitutas da Imunoglobulina/genética , Mieloma Múltiplo/genética , Proliferação de Células , Edição de Genes , Terapia Genética , Humanos , Mieloma Múltiplo/patologia , Mieloma Múltiplo/terapia , RNA Mensageiro/genética , Células Tumorais CultivadasRESUMO
Renal cell carcinomas (RCCs) account for about 90% of renal tumors, and their major histological subtype is ccRCC (clear cell RCC). Increasing evidence has indicated that the tumor microenvironment plays a significant role in the occurrence and development of ccRCC. In this study, we used ESTIMATE and CIBERSORT computational methods to calculate the proportion of immune and stromal components and the rate of TICs (tumor-infiltrating immune cells) in 539 ccRCC samples from The Cancer Genome Atlas database. By examining the intersection of the differentially expressed genes obtained by the protein-protein interaction network and Cox regression analysis, we identified only one overlapping gene: IGLL5 (immunoglobulin lambda-like polypeptide 5). We report that IGLL5 expression is correlated with TICs. Furthermore, our immunoinfiltration analyses revealed that three types of TIC are positively correlated with IGLL5 expression. IGLL5 may have potential as a prognostic biomarker of ccRCC.
Assuntos
Carcinoma de Células Renais/patologia , Cadeias Leves Substitutas da Imunoglobulina/genética , Neoplasias Renais/patologia , Linfócitos do Interstício Tumoral/metabolismo , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/imunologia , Biologia Computacional , Feminino , Humanos , Neoplasias Renais/genética , Neoplasias Renais/imunologia , Masculino , Estadiamento de Neoplasias , Prognóstico , Mapas de Interação de Proteínas , Análise de Sobrevida , Microambiente TumoralRESUMO
B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells, B-1a cells have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues, the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver versus bone marrow environment, reduced IL-7R/STAT5 levels promote immunoglobulin kappa gene recombination at the early pro-B cell stage. As a result, differentiating B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain. This 'alternate pathway' of development enables the production of B cells with self-reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing lineage and selection models of B-1a cell development and explain how these cells acquire their unique properties.
Assuntos
Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , Diferenciação Celular/imunologia , Receptores de Células Precursoras de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Subpopulações de Linfócitos B/metabolismo , Linfócitos B/metabolismo , Medula Óssea/imunologia , Medula Óssea/metabolismo , Diferenciação Celular/genética , Cadeias Leves Substitutas da Imunoglobulina/genética , Cadeias Leves Substitutas da Imunoglobulina/imunologia , Cadeias Leves Substitutas da Imunoglobulina/metabolismo , Fígado/embriologia , Fígado/imunologia , Fígado/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Células Precursoras de Linfócitos B/genética , Receptores de Células Precursoras de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/imunologia , Receptores de Interleucina-7/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/imunologia , Fator de Transcrição STAT5/metabolismoRESUMO
Multiple myeloma (MM) is a disease of copy number variants (CNVs), chromosomal translocations, and single-nucleotide variants (SNVs). To enable integrative studies across these diverse mutation types, we developed a capture-based sequencing platform to detect their occurrence in 465 genes altered in MM and used it to sequence 95 primary tumor-normal pairs to a mean depth of 104×. We detected cases of hyperdiploidy (23%), deletions of 1p (8%), 6q (21%), 8p (17%), 14q (16%), 16q (22%), and 17p (4%), and amplification of 1q (19%). We also detected IGH and MYC translocations near expected frequencies and non-silent SNVs in NRAS (24%), KRAS (21%), FAM46C (17%), TP53 (9%), DIS3 (9%), and BRAF (3%). We discovered frequent mutations in IGLL5 (18%) that were mutually exclusive of RAS mutations and associated with increased risk of disease progression (p = 0.03), suggesting that IGLL5 may be a stratifying biomarker. We identified novel IGLL5/IGH translocations in two samples. We subjected 15 of the pairs to ultra-deep sequencing (1259×) and found that although depth correlated with number of mutations detected (p = 0.001), depth past ~300× added little. The platform provides cost-effective genomic analysis for research and may be useful in individualizing treatment decisions in clinical settings.
Assuntos
Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves Substitutas da Imunoglobulina/genética , Mieloma Múltiplo/genética , Mutação Puntual , Translocação Genética , Biomarcadores Tumorais , Variações do Número de Cópias de DNA , Expressão Gênica , Genes myc , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Estimativa de Kaplan-Meier , Proteínas de Membrana/genética , Polimorfismo de Nucleotídeo Único , PrognósticoRESUMO
Follicular lymphoma (FL) is an indolent B-cell non-Hodgkin lymphoma able to transform into germinal center-type diffuse large B-cell lymphoma. We describe four extraordinary cases of FL, which progressed to TdT+CD20- precursor B-lymphoblastic lymphoma (B-LBL). Fluorescence in situ hybridization analysis showed that all four B-LBLs had acquired a MYC translocation on transformation. Comparative genomic hybridization analysis of one case demonstrated that in addition to 26 numerical aberrations that were shared between the FL and B-LBL, deletion of CDKN2A/B and 17q11, 14q32 amplification, and copy-neutral loss of heterozygosity of 9p were gained in the B-LBL cells. Whole-exome sequencing revealed mutations in FMN2, NEB, and SYNE1 and a nonsense mutation in KMT2D, all shared by the FL and B-LBL, and TNFRSF14, SMARCA2, CCND3 mutations uniquely present in the B-LBL. Remarkably, all four FL-B-LBL pairs expressed IgG. In two B-LBLs, evidence was obtained for ongoing rearrangement of IG light chain variable genes and expression of the surrogate light chain. IGHV mutation analysis showed that all FL-B-LBL pairs harbored identical or near-identical somatic mutations. From the somatic gene alterations found in the IG and non-IG genes, we conclude that the FLs and B-LBLs did not develop in parallel from early t(14;18)-positive IG-unmutated precursors, but that the B-LBLs developed from preexistent FL subclones that accumulated additional genetic damage.
Assuntos
Cadeias Leves Substitutas da Imunoglobulina/genética , Cadeias gama de Imunoglobulina/genética , Linfoma de Células B/genética , Linfoma Folicular/genética , Linfócitos B/patologia , Hibridização Genômica Comparativa , Ciclina D3/genética , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor p16 de Quinase Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p18/genética , Análise Mutacional de DNA , Feminino , Centro Germinativo/patologia , Humanos , Cadeias Leves Substitutas da Imunoglobulina/metabolismo , Cadeias gama de Imunoglobulina/metabolismo , Hibridização in Situ Fluorescente , Linfoma de Células B/patologia , Linfoma Folicular/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Neurofibromina 1/genética , Membro 14 de Receptores do Fator de Necrose Tumoral/genética , Fatores de Transcrição/genética , Translocação Genética , Adulto JovemRESUMO
Precursor-B cell receptor (pre-BCR) signaling represents a crucial checkpoint at the pre-B cell stage. Aberrant pre-BCR signaling is considered as a key factor for B-cell precursor acute lymphoblastic leukemia (BCP-ALL) development. BCP-ALL are believed to be arrested at the pre-BCR checkpoint independent of pre-BCR expression. However, the cellular stage at which BCP-ALL are arrested and whether this relates to expression of the pre-BCR components (IGHM, IGLL1 and VPREB1) is still unclear. Here, we show differential protein expression and copy number variation (CNV) patterns of the pre-BCR components in pediatric BCP-ALL. Moreover, analyzing six BCP-ALL data sets (n = 733), we demonstrate that TCF3-PBX1 ALL express high levels of IGHM, IGLL1 and VPREB1, and are arrested at the pre-B stage. By contrast, ETV6-RUNX1 ALL express low levels of IGHM or VPREB1, and are arrested at the pro-B stage. Irrespective of subtype, ALL with high levels of IGHM, IGLL1 and VPREB1 are arrested at the pre-B stage and correlate with good prognosis in high-risk pediatric BCP-ALL (n = 207). Our findings suggest that BCP-ALL are arrested at different cellular stages, which relates to the expression pattern of the pre-BCR components that could serve as prognostic markers for high-risk pediatric BCP-ALL patients.
Assuntos
Regulação Leucêmica da Expressão Gênica , Doença das Cadeias Pesadas/genética , Cadeias Leves Substitutas da Imunoglobulina/genética , Cadeias mu de Imunoglobulina/genética , Receptores de Células Precursoras de Linfócitos B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Criança , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Variações do Número de Cópias de DNA/genética , Perfilação da Expressão Gênica , Doença das Cadeias Pesadas/metabolismo , Humanos , Cadeias Leves Substitutas da Imunoglobulina/metabolismo , Cadeias mu de Imunoglobulina/metabolismo , Estadiamento de Neoplasias , Proteínas de Fusão Oncogênica/metabolismo , Receptores de Células Precursoras de Linfócitos B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Resultado do TratamentoRESUMO
Copy number variation (CNV) is a newly discovered form of intra-species genetic polymorphism that is defined as deletions or duplications of genome segments ranging from 1 kbp to several Mbp. CNV accounts for the majority of the genetic variation observed in humans (CNV regions cover more than 10% of the human genome); therefore, it may significantly influence both the phenotype and susceptibility to various diseases. Unfortunately, the impact of CNV on a number of diseases, including hepatitis C virus (HCV) infection, remains largely unexplored. Here, we analyzed 421 human genes encoding proteins that have been shown to interact with HCV proteins or genomic RNA (proteins from the HCV-human interactome). We found that 19 of the 421 candidate genes are located in putative CNV regions. For all of these genes, copy numbers were determined for European, Asiatic and African populations using the multiplex ligation-dependent amplification (MLPA) method. As a result, we identified 4 genes, IGLL1, MLLT4, PDPK1, PPP1R13L, for which the CN-genotype ranged from 1 to 6. All of these genes are involved in host-virus interaction; thus, their polymorphism has a potential impact on the development of HCV infection and/or therapy outcome.
Assuntos
Variações do Número de Cópias de DNA , Redes Reguladoras de Genes , Hepacivirus/fisiologia , Hepatite C/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Biologia Computacional/métodos , Hepacivirus/metabolismo , Hepatite C/etnologia , Hepatite C/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Cadeias Leves Substitutas da Imunoglobulina/genética , Cadeias Leves Substitutas da Imunoglobulina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Miosinas/genética , Miosinas/metabolismo , Grupos Raciais/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Virais/metabolismoRESUMO
Substrate binding to Hsp70 chaperones is involved in many biological processes, and the identification of potential substrates is important for a comprehensive understanding of these events. We present a multi-scale pipeline for an accurate, yet efficient prediction of peptides binding to the Hsp70 chaperone BiP by combining sequence-based prediction with molecular docking and MMPBSA calculations. First, we measured the binding of 15mer peptides from known substrate proteins of BiP by peptide array (PA) experiments and performed an accuracy assessment of the PA data by fluorescence anisotropy studies. Several sequence-based prediction models were fitted using this and other peptide binding data. A structure-based position-specific scoring matrix (SB-PSSM) derived solely from structural modeling data forms the core of all models. The matrix elements are based on a combination of binding energy estimations, molecular dynamics simulations, and analysis of the BiP binding site, which led to new insights into the peptide binding specificities of the chaperone. Using this SB-PSSM, peptide binders could be predicted with high selectivity even without training of the model on experimental data. Additional training further increased the prediction accuracies. Subsequent molecular docking (DynaDock) and MMGBSA/MMPBSA-based binding affinity estimations for predicted binders allowed the identification of the correct binding mode of the peptides as well as the calculation of nearly quantitative binding affinities. The general concept behind the developed multi-scale pipeline can readily be applied to other protein-peptide complexes with linearly bound peptides, for which sufficient experimental binding data for the training of classical sequence-based prediction models is not available. Proteins 2016; 84:1390-1407. © 2016 Wiley Periodicals, Inc.
Assuntos
Proteínas de Transporte/química , Proteínas de Choque Térmico/química , Cadeias Leves Substitutas da Imunoglobulina/química , Peptídeos/química , Fator A de Crescimento do Endotélio Vascular/química , Sequência de Aminoácidos , Anisotropia , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Chaperona BiP do Retículo Endoplasmático , Corantes Fluorescentes/química , Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Cadeias Leves Substitutas da Imunoglobulina/genética , Cadeias Leves Substitutas da Imunoglobulina/metabolismo , Isoquinolinas/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeos/genética , Peptídeos/metabolismo , Análise Serial de Proteínas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Termodinâmica , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The proliferation of pre-B cells is known to further increase the clonal diversity of B cells at the stage of pre-B cells by allowing the same rearranged heavy chains to combine with differently rearranged light chains in a subsequent developmental stage. Crlz-1 (charged amino acid-rich leucine zipper-1) was found to control this proliferation of pre-B cells by working as a Wnt (wingless-related mouse mammary tumor virus integration site) target gene in these cells. Mechanistically, Crlz-1 protein functioned by mobilizing cytoplasmic CBFß (core binding factor ß) into the nucleus to allow Runx (runt-related transcription factor)/CBFß heterodimerization. Runx/CBFß then turned on its target genes such as EBF (early B cell factor), VpreB, and λ5 and thereby pre-B cell receptor signaling, leading to the expression of cyclins D2 and D3 Actually, the proliferative function of Crlz-1 was demonstrated by not only Crlz-1 or ß-catenin knockdown but also Crlz-1 overexpression. Furthermore, the mechanistic view that the proliferative function of Crlz-1 is caused by relaying Wnt/ß-catenin to pre-B cell receptor signaling pathways through the regulation of Runx/CBFß heterodimerization was also verified by employing niclosamide, XAV939, and LiCl as Wnt inhibitors and activator, respectively.
Assuntos
Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Subunidade beta de Fator de Ligação ao Core/metabolismo , Proteínas de Ligação a DNA/metabolismo , Cadeias Leves Substitutas da Imunoglobulina/genética , Proteínas do Tecido Nervoso/metabolismo , Células Precursoras de Linfócitos B/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células Cultivadas , Subunidades alfa de Fatores de Ligação ao Core/genética , Subunidade beta de Fator de Ligação ao Core/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Compostos Heterocíclicos com 3 Anéis/farmacologia , Cadeias Leves Substitutas da Imunoglobulina/metabolismo , Cloreto de Lítio/farmacologia , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Camundongos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Niclosamida/farmacologia , Receptores de Células Precursoras de Linfócitos B/genética , Receptores de Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/efeitos dos fármacos , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Regulação para Cima , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética , beta Catenina/antagonistas & inibidores , beta Catenina/genética , beta Catenina/metabolismoRESUMO
TCF3-HLF-positive acute lymphoblastic leukemia (ALL) is currently incurable. Using an integrated approach, we uncovered distinct mutation, gene expression and drug response profiles in TCF3-HLF-positive and treatment-responsive TCF3-PBX1-positive ALL. We identified recurrent intragenic deletions of PAX5 or VPREB1 in constellation with the fusion of TCF3 and HLF. Moreover somatic mutations in the non-translocated allele of TCF3 and a reduction of PAX5 gene dosage in TCF3-HLF ALL suggest cooperation within a restricted genetic context. The enrichment for stem cell and myeloid features in the TCF3-HLF signature may reflect reprogramming by TCF3-HLF of a lymphoid-committed cell of origin toward a hybrid, drug-resistant hematopoietic state. Drug response profiling of matched patient-derived xenografts revealed a distinct profile for TCF3-HLF ALL with resistance to conventional chemotherapeutics but sensitivity to glucocorticoids, anthracyclines and agents in clinical development. Striking on-target sensitivity was achieved with the BCL2-specific inhibitor venetoclax (ABT-199). This integrated approach thus provides alternative treatment options for this deadly disease.
Assuntos
Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Técnicas de Cocultura , Estudos de Coortes , Análise Mutacional de DNA , Resistencia a Medicamentos Antineoplásicos , Feminino , Expressão Gênica , Estudos de Associação Genética , Genômica , Humanos , Cadeias Leves Substitutas da Imunoglobulina/genética , Concentração Inibidora 50 , Estimativa de Kaplan-Meier , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Proteínas de Fusão Oncogênica/metabolismo , Fator de Transcrição PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Deleção de Sequência , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Random recombination of antibody heavy- and light-chain genes results in a diverse B-cell receptor (BCR) repertoire including self-reactive BCRs. However, tolerance mechanisms that prevent the development of self-reactive B cells remain incompletely understood. The absence of the surrogate light chain, which assembles with antibody heavy chain forming a pre-BCR, leads to production of antinuclear antibodies (ANAs). Here we show that the naive follicular B-cell pool is enriched for cells expressing prototypic ANA heavy chains in these mice in a non-autoimmune background with a broad antibody repertoire. This results in the spontaneous formation of T-cell-dependent germinal centres that are enriched with B cells expressing prototypic ANA heavy chains. However, peripheral tolerance appears maintained by selection thresholds on cells entering the memory B-cell and plasma cell pools, as exemplified by the exclusion of cells expressing the intrinsically self-reactive V(H)81X from both pools.
Assuntos
Anticorpos Antinucleares/metabolismo , Linfócitos B/fisiologia , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves Substitutas da Imunoglobulina/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Antibacterianos , Anticorpos Antinucleares/genética , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves Substitutas da Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos B/genética , Baço/citologia , Linfócitos TRESUMO
The adverse effects of alcohol consumption during pregnancy are known, but the molecular events that lead to the phenotypic characteristics are unclear. To unravel the molecular mechanisms, we have used a mouse model of gestational ethanol exposure, which is based on maternal ad libitum ingestion of 10% (v/v) ethanol for the first 8 days of gestation (GD 0.5-8.5). Early neurulation takes place by the end of this period, which is equivalent to the developmental stage early in the fourth week post-fertilization in human. During this exposure period, dynamic epigenetic reprogramming takes place and the embryo is vulnerable to the effects of environmental factors. Thus, we hypothesize that early ethanol exposure disrupts the epigenetic reprogramming of the embryo, which leads to alterations in gene regulation and life-long changes in brain structure and function. Genome-wide analysis of gene expression in the mouse hippocampus revealed altered expression of 23 genes and three miRNAs in ethanol-exposed, adolescent offspring at postnatal day (P) 28. We confirmed this result by using two other tissues, where three candidate genes are known to express actively. Interestingly, we found a similar trend of upregulated gene expression in bone marrow and main olfactory epithelium. In addition, we observed altered DNA methylation in the CpG islands upstream of the candidate genes in the hippocampus. Our MRI study revealed asymmetry of brain structures in ethanol-exposed adult offspring (P60): we detected ethanol-induced enlargement of the left hippocampus and decreased volume of the left olfactory bulb. Our study indicates that ethanol exposure in early gestation can cause changes in DNA methylation, gene expression, and brain structure of offspring. Furthermore, the results support our hypothesis of early epigenetic origin of alcohol-induced disorders: changes in gene regulation may have already taken place in embryonic stem cells and therefore can be seen in different tissue types later in life.
Assuntos
Consumo de Bebidas Alcoólicas/metabolismo , Metilação de DNA/efeitos dos fármacos , Epigênese Genética , Etanol/farmacologia , Hipocampo/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Consumo de Bebidas Alcoólicas/genética , Consumo de Bebidas Alcoólicas/patologia , Animais , Medula Óssea/efeitos dos fármacos , Ilhas de CpG , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Hipocampo/patologia , Histonas/genética , Histonas/metabolismo , Humanos , Cadeias Leves Substitutas da Imunoglobulina/genética , Cadeias Leves Substitutas da Imunoglobulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Neurulação/efeitos dos fármacos , Neurulação/genética , Bulbo Olfatório/efeitos dos fármacos , Bulbo Olfatório/patologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/genética , Efeitos Tardios da Exposição Pré-Natal/patologia , Receptores Odorantes/genética , Receptores Odorantes/metabolismoRESUMO
Selection of the primary antibody repertoire takes place in pro-/pre-B cells, and subsequently in immature and transitional B cells. At the first checkpoint, µ heavy (µH) chains assemble with surrogate light (SL) chain into a precursor B-cell receptor. In mice lacking SL chain, µH chain selection is impaired, and serum autoantibody levels are elevated. However, whether the development of autoantibody-producing cells is due to an inability of the resultant B-cell receptors to induce central and/or peripheral B-cell tolerance or other factors is unknown. Here, we show that receptor editing is defective, and that a higher proportion of BM immature B cells are prone to undergoing apoptosis. Furthermore, transitional B cells are also more prone to undergoing apoptosis, with a stronger selection pressure to enter the follicular B-cell pool. Those that enter the marginal zone (MZ) B-cell pool escape selection and survive, possibly due to the B-lymphopenia and elevated levels of B-cell activating factor. Moreover, the MZ B cells are responsible for the elevated IgM anti-dsDNA antibody levels detected in these mice. Thus, the SL chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by MZ B cells.
Assuntos
Anticorpos Antinucleares/biossíntese , Linfócitos B/imunologia , Tolerância Imunológica/imunologia , Cadeias Leves Substitutas da Imunoglobulina/genética , Animais , Anticorpos Antinucleares/imunologia , Formação de Anticorpos/genética , Apoptose/imunologia , Autoanticorpos/biossíntese , Autoanticorpos/sangue , Fator Ativador de Células B/metabolismo , Receptor do Fator Ativador de Células B/metabolismo , Proteínas de Homeodomínio/genética , Cadeias Leves Substitutas da Imunoglobulina/imunologia , Imunoglobulina M/imunologia , Cadeias mu de Imunoglobulina/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos B/biossínteseRESUMO
CD27, a member of the TNFR superfamily, is used to identify human memory B cells. Nonetheless, CD27(+) B cells are present in patients with HIGM1 syndrome who are unable to generate GCs or memory B cells. CD27(+)IgD(+) fetal B cells are present in umbilical cord blood, and CD27 may also be a marker of the human B1-like B cells. To define the origin of naïve CD27(+)IgD(+) human B cells, we studied B cell development in both fetal and adult tissues. In human FL, most CD19(+) cells coexpressed CD10, a marker of human developing B cells. Some CD19(+)CD10(+) B cells expressed CD27, and these fetal CD27(+) cells were present in the pro-B, pre-B, and immature/transitional B cell compartments. Lower frequencies of phenotypically identical cells were also identified in adult BM. CD27(+) pro-B, pre-B, and immature/transitional B cells expressed recombination activating gene-1, terminal deoxynucleotidyl transferase and Vpre-B mRNA comparably to their CD27(-) counterparts. CD27(+) and CD27(-) developing B cells showed similar Ig heavy chain gene usage with low levels of mutations, suggesting that CD27(+) developing B cells are distinct from mutated memory B cells. Despite these similarities, CD27(+) developing B cells differed from CD27(-) developing B cells by their increased expression of LIN28B, a transcription factor associated with the fetal lymphoid lineages of mice. Furthermore, CD27(+) pro-B cells efficiently generated IgM(+)IgD(+) immature/transitional B cells in vitro. Our observations suggest that CD27 expression during B cell development identifies a physiologic state or lineage for human B cell development distinct from the memory B cell compartment.
Assuntos
Linhagem da Célula , Proteínas de Ligação a DNA/análise , Feto/imunologia , Células Precursoras de Linfócitos B/fisiologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/análise , Adolescente , Adulto , Antígenos CD19/análise , DNA Nucleotidilexotransferase/genética , Feminino , Proteínas de Homeodomínio/genética , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves Substitutas da Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Masculino , Neprilisina/análise , Mutação Puntual , Células Precursoras de Linfócitos B/química , Células Precursoras de Linfócitos B/imunologia , RNA Mensageiro/análise , Proteínas de Ligação a RNARESUMO
Fetal cattle B-cell development proceeds via a pre-B cell stage that is characterized by the expression of surrogate light chain and recombination activation genes. In this paper, we identify a new member of bovine pre-B lymphocyte genes, VPREB2. Using RT-qPCR, we assess the expression of VPREB2 and three other surrogate light chain genes as well as RAG1 and RAG2 in fetal and adult cattle tissues. The absence of VPREB1, IGLL1, RAG1 and RAG2 expression in adult tissues and the lack of B-lymphoid differentiation in adult bone marrow - OP9 stromal cell co-culture, suggest a decline of B lymphopoiesis in adult cattle. The marked differences in the expression profiles of VPREB2 and VPREB3 in comparison to those of VPREB1, IGLL1 and RAGs suggest that the biological roles of VPREB2 and VPREB3 are unrelated to the pre-B cells.
Assuntos
Linfócitos B/fisiologia , Feto/citologia , Expressão Gênica , Cadeias Leves Substitutas da Imunoglobulina/genética , Linfopoese/genética , Animais , Linfócitos B/metabolismo , Biomarcadores/metabolismo , Medula Óssea/metabolismo , Bovinos , Células Cultivadas , Clonagem Molecular , Técnicas de Cocultura , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Cadeias Leves Substitutas da Imunoglobulina/metabolismo , Linfonodos/metabolismo , Especificidade de Órgãos , Filogenia , Análise de Sequência de DNARESUMO
Induced pluripotent stem (iPS) cells offer a unique potential for understanding the molecular basis of disease and development. Here we have generated several human iPS cell lines, and we describe their pluripotent phenotype and ability to differentiate into erythroid cells, monocytes, and endothelial cells. More significantly, however, when these iPS cells were differentiated under conditions that promote lympho-hematopoiesis from human embryonic stem cells, we observed the formation of pre-B cells. These cells were CD45(+)CD19(+)CD10(+) and were positive for transcripts Pax5, IL7αR, λ-like, and VpreB receptor. Although they were negative for surface IgM and CD5 expression, iPS-derived CD45(+)CD19(+) cells also exhibited multiple genomic D-J(H) rearrangements, which supports a pre-B-cell identity. We therefore have been able to demonstrate, for the first time, that human iPS cells are able to undergo hematopoiesis that contributes to the B-cell lymphoid lineage.