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1.
Cell ; 186(6): 1144-1161.e18, 2023 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-36868219

RESUMO

Germinal centers (GCs) that form within lymphoid follicles during antibody responses are sites of massive cell death. Tingible body macrophages (TBMs) are tasked with apoptotic cell clearance to prevent secondary necrosis and autoimmune activation by intracellular self antigens. We show by multiple redundant and complementary methods that TBMs derive from a lymph node-resident, CD169-lineage, CSF1R-blockade-resistant precursor that is prepositioned in the follicle. Non-migratory TBMs use cytoplasmic processes to chase and capture migrating dead cell fragments using a "lazy" search strategy. Follicular macrophages activated by the presence of nearby apoptotic cells can mature into TBMs in the absence of GCs. Single-cell transcriptomics identified a TBM cell cluster in immunized lymph nodes which upregulated genes involved in apoptotic cell clearance. Thus, apoptotic B cells in early GCs trigger activation and maturation of follicular macrophages into classical TBMs to clear apoptotic debris and prevent antibody-mediated autoimmune diseases.


Assuntos
Centro Germinativo , Linfonodos , Macrófagos , Apoptose , Linfócitos B , Linfonodos/citologia , Macrófagos/citologia , Macrófagos/metabolismo
2.
Cell ; 184(5): 1330-1347.e13, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33636130

RESUMO

Osteoclasts are large multinucleated bone-resorbing cells formed by the fusion of monocyte/macrophage-derived precursors that are thought to undergo apoptosis once resorption is complete. Here, by intravital imaging, we reveal that RANKL-stimulated osteoclasts have an alternative cell fate in which they fission into daughter cells called osteomorphs. Inhibiting RANKL blocked this cellular recycling and resulted in osteomorph accumulation. Single-cell RNA sequencing showed that osteomorphs are transcriptionally distinct from osteoclasts and macrophages and express a number of non-canonical osteoclast genes that are associated with structural and functional bone phenotypes when deleted in mice. Furthermore, genetic variation in human orthologs of osteomorph genes causes monogenic skeletal disorders and associates with bone mineral density, a polygenetic skeletal trait. Thus, osteoclasts recycle via osteomorphs, a cell type involved in the regulation of bone resorption that may be targeted for the treatment of skeletal diseases.


Assuntos
Reabsorção Óssea/patologia , Osteoclastos/patologia , Ligante RANK/metabolismo , Animais , Apoptose , Reabsorção Óssea/metabolismo , Fusão Celular , Células Cultivadas , Humanos , Macrófagos/citologia , Camundongos , Osteocondrodisplasias/tratamento farmacológico , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/patologia , Osteoclastos/metabolismo , Transdução de Sinais
4.
Immunity ; 42(5): 890-902, 2015 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-25979420

RESUMO

The mechanistic links between genetic variation and autoantibody production in autoimmune disease remain obscure. Autoimmune lymphoproliferative syndrome (ALPS) is caused by inactivating mutations in FAS or FASL, with autoantibodies thought to arise through failure of FAS-mediated removal of self-reactive germinal center (GC) B cells. Here we show that FAS is in fact not required for this process. Instead, FAS inactivation led to accumulation of a population of unconventional GC B cells that underwent somatic hypermutation, survived despite losing antigen reactivity, and differentiated into a large population of plasma cells that included autoantibody-secreting clones. IgE(+) plasma cell numbers, in particular, increased after FAS inactivation and a major cohort of ALPS-affected patients were found to have hyper-IgE. We propose that these previously unidentified cells, designated "rogue GC B cells," are a major driver of autoantibody production and provide a mechanistic explanation for the linked production of IgE and autoantibodies in autoimmune disease.


Assuntos
Autoanticorpos/imunologia , Linfócitos B/citologia , Centro Germinativo/citologia , Centro Germinativo/imunologia , Imunoglobulina E/imunologia , Receptor fas/imunologia , Animais , Autoanticorpos/biossíntese , Linfócitos B/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Humanos , Imunoglobulina E/biossíntese , Camundongos , Reação em Cadeia da Polimerase , Receptor fas/deficiência , Receptor fas/metabolismo
5.
Immunity ; 42(4): 704-18, 2015 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-25840682

RESUMO

B helper follicular T (Tfh) cells are critical for long-term humoral immunity. However, it remains unclear how these cells are recruited and contribute to secondary immune responses. Here we show that primary Tfh cells segregate into follicular mantle (FM) and germinal center (GC) subpopulations that display distinct gene expression signatures. Restriction of the primary Tfh cell subpopulation in the GC was mediated by downregulation of chemotactic receptor EBI2. Following collapse of the GC, memory T cells persisted in the outer follicle where they scanned CD169(+) subcapsular sinus macrophages. Reactivation and intrafollicular expansion of these follicular memory T cells in the subcapsular region was followed by their extrafollicular dissemination via the lymphatic flow. These data suggest that Tfh cells integrate their antigen-experience history to focus T cell help within the GC during primary responses but act rapidly to provide systemic T cell help after re-exposure to the antigen.


Assuntos
Linfócitos B/citologia , Linhagem da Célula/imunologia , Centro Germinativo/citologia , Imunidade Humoral , Linfócitos T Auxiliares-Indutores/citologia , Animais , Linfócitos B/imunologia , Diferenciação Celular , Linhagem da Célula/genética , Movimento Celular/imunologia , Proliferação de Células , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Centro Germinativo/imunologia , Memória Imunológica , Camundongos , Camundongos Knockout , Cultura Primária de Células , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/imunologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Transdução de Sinais , Linfócitos T Auxiliares-Indutores/imunologia
6.
Trends Immunol ; 40(1): 35-48, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30502023

RESUMO

Subcapsular sinus (SCS) macrophages are strategically positioned at the lymph-tissue interface in the lymph node to trap and present antigen to B cells. Recent murine data has shown that SCS macrophages also prevent the systemic spread of lymph-borne pathogens and are capable of activating a diverse range of innate effector and adaptive memory cells, including follicular memory T cells and memory B cells (Bmems), that are either pre-positioned or rapidly recruited to the subcapsular niche following infection and inflammation. Furthermore, Bmems are rapidly reactivated to differentiate into plasma cells in subcapsular proliferative foci (SPF). Thus, understanding how SCS macrophages coordinate both innate and adaptive memory responses in the subcapsular niche can provide new opportunities to bolster immunity against pathogens and cancer.


Assuntos
Imunidade Adaptativa/imunologia , Imunidade Inata/imunologia , Linfonodos/imunologia , Macrófagos/imunologia , Animais , Camundongos
7.
Blood ; 134(1): 30-43, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31023703

RESUMO

The era of targeted therapies has seen significant improvements in depth of response, progression-free survival, and overall survival for patients with multiple myeloma. Despite these improvements in clinical outcome, patients inevitably relapse and require further treatment. Drug-resistant dormant myeloma cells that reside in specific niches within the skeleton are considered a basis of disease relapse but remain elusive and difficult to study. Here, we developed a method to sequence the transcriptome of individual dormant myeloma cells from the bones of tumor-bearing mice. Our analyses show that dormant myeloma cells express a distinct transcriptome signature enriched for immune genes and, unexpectedly, genes associated with myeloid cell differentiation. These genes were switched on by coculture with osteoblastic cells. Targeting AXL, a gene highly expressed by dormant cells, using small-molecule inhibitors released cells from dormancy and promoted their proliferation. Analysis of the expression of AXL and coregulated genes in human cohorts showed that healthy human controls and patients with monoclonal gammopathy of uncertain significance expressed higher levels of the dormancy signature genes than patients with multiple myeloma. Furthermore, in patients with multiple myeloma, the expression of this myeloid transcriptome signature translated into a twofold increase in overall survival, indicating that this dormancy signature may be a marker of disease progression. Thus, engagement of myeloma cells with the osteoblastic niche induces expression of a suite of myeloid genes that predicts disease progression and that comprises potential drug targets to eradicate dormant myeloma cells.


Assuntos
Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Recidiva Local de Neoplasia/genética , Células-Tronco Neoplásicas/patologia , Nicho de Células-Tronco/genética , Animais , Humanos , Camundongos , Recidiva Local de Neoplasia/patologia , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Transcriptoma , Receptor Tirosina Quinase Axl
8.
Breast Cancer Res ; 17: 83, 2015 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-26070602

RESUMO

INTRODUCTION: The study of mammalian development has offered many insights into the molecular aetiology of cancer. We previously used analysis of mammary morphogenesis to discover a critical role for GATA-3 in mammary developmental and carcinogenesis. In recent years an important role for microRNAs (miRNAs) in a myriad of cellular processes in development and in oncogenesis has emerged. METHODS: microRNA profiling was conducted on stromal and epithelial cellular subsets microdissected from the pubertal mouse mammary gland. miR-184 was reactivated by transient or stable overexpression in breast cancer cell lines and examined using a series of in vitro (proliferation, tumour-sphere and protein synthesis) assays. Orthotopic xenografts of breast cancer cells were used to assess the effect of miR-184 on tumourigenesis as well as distant metastasis. Interactions between miR-184 and its putative targets were assessed by quantitative PCR, microarray, bioinformatics and 3' untranslated region Luciferase reporter assay. The methylation status of primary patient samples was determined by MBD-Cap sequencing. Lastly, the clinical prognostic significance of miR-184 putative targets was assessed using publicly available datasets. RESULTS: A large number of microRNA were restricted in their expression to specific tissue subsets. MicroRNA-184 (miR-184) was exclusively expressed in epithelial cells and markedly upregulated during differentiation of the proliferative, invasive cells of the pubertal terminal end bud (TEB) into ductal epithelial cells in vivo. miR-184 expression was silenced in mouse tumour models compared to non-transformed epithelium and in a majority of breast cancer cell line models. Ectopic reactivation of miR-184 inhibited the proliferation and self-renewal of triple negative breast cancer (TNBC) cell lines in vitro and delayed primary tumour formation and reduced metastatic burden in vivo. Gene expression studies uncovered multi-factorial regulation of genes in the AKT/mTORC1 pathway by miR-184. In clinical breast cancer tissues, expression of miR-184 is lost in primary TNBCs while the miR-184 promoter is methylated in a subset of lymph node metastases from TNBC patients. CONCLUSIONS: These studies elucidate a new layer of regulation in the PI3K/AKT/mTOR pathway with relevance to mammary development and tumour progression and identify miR-184 as a putative breast tumour suppressor.


Assuntos
Neoplasias da Mama/genética , Perfilação da Expressão Gênica , Genes Supressores de Tumor , Glândulas Mamárias Animais/metabolismo , MicroRNAs/genética , Maturidade Sexual/genética , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Análise por Conglomerados , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Camundongos , Metástase Neoplásica , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Serina-Treonina Quinases TOR/metabolismo
9.
Front Immunol ; 9: 1553, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30022984

RESUMO

Single-cell RNA sequencing (scRNA-Seq) is transforming our ability to characterize cells, particularly rare cells that are often overlooked in bulk population analytical approaches. This has lead to the discovery of new cell types and cellular states that echo the underlying heterogeneity and plasticity in the immune system. Technologies for the capture, sequencing, and bioinformatic analysis of single cells are rapidly improving, and scRNA-Seq is now becoming much more accessible to non-specialized laboratories. Here, we describe our experiences in adopting scRNA-Seq to the study of rare immune cells in their microanatomical niches.

10.
Nat Commun ; 9(1): 3372, 2018 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-30135429

RESUMO

Vaccine-induced immunity depends on the generation of memory B cells (MBC). However, where and how MBCs are reactivated to make neutralising antibodies remain unknown. Here we show that MBCs are prepositioned in a subcapsular niche in lymph nodes where, upon reactivation by antigen, they rapidly proliferate and differentiate into antibody-secreting plasma cells in the subcapsular proliferative foci (SPF). This novel structure is enriched for signals provided by T follicular helper cells and antigen-presenting subcapsular sinus macrophages. Compared with contemporaneous secondary germinal centres, SPF have distinct single-cell molecular signature, cell migration pattern and plasma cell output. Moreover, SPF are found both in human and mouse lymph nodes, suggesting that they are conserved throughout mammalian evolution. Our data thus reveal that SPF is a seat of immunological memory that may be exploited to rapidly mobilise secondary antibody responses and improve vaccine efficacy.


Assuntos
Linfócitos B/metabolismo , Linfonodos/metabolismo , Adenina/análogos & derivados , Animais , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Citometria de Fluxo , Humanos , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Teóricos , Piperidinas , Pirazóis/farmacologia , Pirimidinas/farmacologia , Tamoxifeno/farmacologia
11.
J Exp Med ; 215(8): 2073-2095, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-30018075

RESUMO

Gain-of-function (GOF) mutations in PIK3CD, encoding the p110δ subunit of phosphatidylinositide 3-kinase (PI3K), cause a primary immunodeficiency. Affected individuals display impaired humoral immune responses following infection or immunization. To establish mechanisms underlying these immune defects, we studied a large cohort of patients with PIK3CD GOF mutations and established a novel mouse model using CRISPR/Cas9-mediated gene editing to introduce a common pathogenic mutation in Pik3cd In both species, hyperactive PI3K severely affected B cell development and differentiation in the bone marrow and the periphery. Furthermore, PI3K GOF B cells exhibited intrinsic defects in class-switch recombination (CSR) due to impaired induction of activation-induced cytidine deaminase (AID) and failure to acquire a plasmablast gene signature and phenotype. Importantly, defects in CSR, AID expression, and Ig secretion were restored by leniolisib, a specific p110δ inhibitor. Our findings reveal key roles for balanced PI3K signaling in B cell development and long-lived humoral immunity and memory and establish the validity of treating affected individuals with p110δ inhibitors.


Assuntos
Linfócitos B/citologia , Linfócitos B/imunologia , Classe I de Fosfatidilinositol 3-Quinases/genética , Mutação em Linhagem Germinativa/genética , Fosfatidilinositol 3-Quinases/genética , Animais , Afinidade de Anticorpos/imunologia , Células da Medula Óssea/citologia , Diferenciação Celular , Proliferação de Células , Criança , Mutação com Ganho de Função/genética , Humanos , Switching de Imunoglobulina , Imunoglobulinas/metabolismo , Interleucinas/farmacologia , Camundongos , Modelos Animais , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Plasmócitos/metabolismo , Transdução de Sinais
12.
J Exp Med ; 213(8): 1589-608, 2016 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-27401342

RESUMO

Naive CD4(+) T cells differentiate into specific effector subsets-Th1, Th2, Th17, and T follicular helper (Tfh)-that provide immunity against pathogen infection. The signaling pathways involved in generating these effector cells are partially known. However, the effects of mutations underlying human primary immunodeficiencies on these processes, and how they compromise specific immune responses, remain unresolved. By studying individuals with mutations in key signaling pathways, we identified nonredundant pathways regulating human CD4(+) T cell differentiation in vitro. IL12Rß1/TYK2 and IFN-γR/STAT1 function in a feed-forward loop to induce Th1 cells, whereas IL-21/IL-21R/STAT3 signaling is required for Th17, Tfh, and IL-10-secreting cells. IL12Rß1/TYK2 and NEMO are also required for Th17 induction. Strikingly, gain-of-function STAT1 mutations recapitulated the impact of dominant-negative STAT3 mutations on Tfh and Th17 cells, revealing a putative inhibitory effect of hypermorphic STAT1 over STAT3. These findings provide mechanistic insight into the requirements for human T cell effector function, and explain clinical manifestations of these immunodeficient conditions. Furthermore, they identify molecules that could be targeted to modulate CD4(+) T cell effector function in the settings of infection, vaccination, or immune dysregulation.


Assuntos
Diferenciação Celular/imunologia , Células Th1/imunologia , Células Th17/imunologia , Células Th2/imunologia , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Diferenciação Celular/genética , Feminino , Humanos , Interleucina-10/genética , Interleucina-10/imunologia , Masculino , Mutação , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Células Th1/citologia , Células Th17/citologia
13.
Cancer Discov ; 5(1): 35-42, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25312016

RESUMO

UNLABELLED: Recent clinical trials have shown that bisphosphonate drugs improve breast cancer patient survival independent of their antiresorptive effects on the skeleton. However, because bisphosphonates bind rapidly to bone mineral, the exact mechanisms of their antitumor action, particularly on cells outside of bone, remain unknown. Here, we used real-time intravital two-photon microscopy to show extensive leakage of fluorescent bisphosphonate from the vasculature in 4T1 mouse mammary tumors, where it initially binds to areas of small, granular microcalcifications that are engulfed by tumor-associated macrophages (TAM), but not tumor cells. Importantly, we also observed uptake of radiolabeled bisphosphonate in the primary breast tumor of a patient and showed the resected tumor to be infiltrated with TAMs and to contain similar granular microcalcifications. These data represent the first compelling in vivo evidence that bisphosphonates can target cells in tumors outside the skeleton and that their antitumor activity is likely to be mediated via TAMs. SIGNIFICANCE: Bisphosphonates are assumed to act solely in bone. However, mouse models and clinical trials show that they have surprising antitumor effects outside bone. We provide unequivocal evidence that bisphosphonates target TAMs, but not tumor cells, to exert their extraskeletal effects, offering a rationale for use in patients with early disease.


Assuntos
Conservadores da Densidade Óssea/metabolismo , Difosfonatos/metabolismo , Macrófagos/metabolismo , Neoplasias/diagnóstico , Tomografia Computadorizada de Emissão de Fóton Único , Tomografia Computadorizada por Raios X , Animais , Conservadores da Densidade Óssea/uso terapêutico , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Calcinose , Carbocianinas , Difosfonatos/uso terapêutico , Modelos Animais de Doenças , Feminino , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Pessoa de Meia-Idade , Gradação de Tumores , Invasividade Neoplásica , Neoplasias/tratamento farmacológico , Fagocitose/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
14.
PLoS One ; 6(2): e17036, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21347226

RESUMO

Approximately 10% of cancers use recombination-mediated Alternative Lengthening of Telomeres (ALT) instead of telomerase to prevent telomere shortening. A characteristic of cells that utilize ALT is the presence of ALT-associated PML nuclear bodies (APBs) containing (TTAGGG)n DNA, telomere binding proteins, DNA recombination proteins, and heterochromatin protein 1 (HP1). The function of APBs is unknown and it is possible that they are functionally heterogeneous. Most ALT cells lack functional p53, and restoration of the p53/p21 pathway in these cells results in growth arrest/senescence and a substantial increase in the number of large APBs that is dependent on two HP1 isoforms, HP1α and HP1γ. Here we investigated the mechanism of HP1-mediated APB formation, and found that histone chaperones, HIRA and ASF1a, are present in APBs following activation of the p53/p21 pathway in ALT cells. HIRA and ASF1a were also found to colocalize inside PML bodies in normal fibroblasts approaching senescence, providing evidence for the existence of a senescence-associated ASF1a/HIRA complex inside PML bodies, consistent with a role for these proteins in induction of senescence in both normal and ALT cells. Moreover, knockdown of HIRA but not ASF1a significantly reduced p53-mediated induction of large APBs, with a concomitant reduction of large HP1 foci. We conclude that HIRA, in addition to its physical and functional association with ASF1a, plays a unique, ASF1a-independent role, which is required for the localization of HP1 to PML bodies and thus for APB formation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Chaperonas de Histonas/metabolismo , Homeostase do Telômero , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Nucléolo Celular/metabolismo , Senescência Celular , Homólogo 5 da Proteína Cromobox , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Chaperonas de Histonas/deficiência , Chaperonas de Histonas/genética , Humanos , Chaperonas Moleculares , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/metabolismo
15.
Cancer Res ; 71(11): 4002-14, 2011 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-21632555

RESUMO

Hedgehog (Hh) signaling plays an important role in several malignancies but its clinical significance in breast cancer is unclear. In a cohort of 279 patients with invasive ductal carcinoma of the breast, expression of Hh ligand was significantly associated with increased risk of metastasis, breast cancer-specific death, and a basal-like phenotype. A paracrine signature, encompassing high epithelial Hh ligand and high stromal Gli1, was an independent predictor for overall survival in multivariate analysis. In 2 independent histological progression series (n = 301), Hh expression increased with atypia. Hh ligand overexpression in a mouse model of basal breast cancer increased growth, induced a poorly differentiated phenotype, accelerated metastasis, and reduced survival. A stromal requirement for these effects was supported by the lack of similar Hh-mediated changes in vitro, and by stromal-specific expression of Hh target genes in vivo. Furthermore, inhibition of Hh ligand with a monoclonal antibody (5E1) inhibited tumor growth and metastasis. These data suggest that epithelial-stromal Hh signaling, driven by ligand expression in carcinoma cells, promotes breast cancer growth and metastasis. Blockade of Hh signaling to peritumoral stromal cells may represent a novel therapeutic approach in some basal-like breast cancers.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas Hedgehog/biossíntese , Animais , Linhagem Celular Tumoral , Estudos de Coortes , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Prognóstico , Transdução de Sinais , Células Estromais/metabolismo , Células Estromais/patologia
16.
Nat Med ; 16(10): 1134-40, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20871609

RESUMO

Inactivation of the p53 tumor suppressor pathway allows cell survival in times of stress and occurs in many human cancers; however, normal embryonic stem cells and some cancers such as neuroblastoma maintain wild-type human TP53 and mouse Trp53 (referred to collectively as p53 herein). Here we describe a miRNA, miR-380-5p, that represses p53 expression via a conserved sequence in the p53 3' untranslated region (UTR). miR-380-5p is highly expressed in mouse embryonic stem cells and neuroblastomas, and high expression correlates with poor outcome in neuroblastomas with neuroblastoma derived v-myc myelocytomatosis viral-related oncogene (MYCN) amplification. miR-380 overexpression cooperates with activated HRAS oncoprotein to transform primary cells, block oncogene-induced senescence and form tumors in mice. Conversely, inhibition of endogenous miR-380-5p in embryonic stem or neuroblastoma cells results in induction of p53, and extensive apoptotic cell death. In vivo delivery of a miR-380-5p antagonist decreases tumor size in an orthotopic mouse model of neuroblastoma. We demonstrate a new mechanism of p53 regulation in cancer and stem cells and uncover a potential therapeutic target for neuroblastoma.


Assuntos
Amplificação de Genes , MicroRNAs/fisiologia , Neuroblastoma/patologia , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Proteína Supressora de Tumor p53/antagonistas & inibidores , Regiões 3' não Traduzidas , Animais , Apoptose , Sítios de Ligação , Dano ao DNA , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/genética , Oncogenes , Proteína Supressora de Tumor p53/fisiologia
17.
J Cell Biol ; 185(5): 797-810, 2009 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-19468068

RESUMO

Alternative lengthening of telomeres (ALT) is a recombination-mediated process that maintains telomeres in telomerase-negative cancer cells. In asynchronously dividing ALT-positive cell populations, a small fraction of the cells have ALT-associated promyelocytic leukemia nuclear bodies (APBs), which contain (TTAGGG)n DNA and telomere-binding proteins. We found that restoring p53 function in ALT cells caused p21 up-regulation, growth arrest/senescence, and a large increase in cells containing APBs. Knockdown of p21 significantly reduced p53-mediated induction of APBs. Moreover, we found that heterochromatin protein 1 (HP1) is present in APBs, and knockdown of HP1alpha and/or HP1gamma prevented p53-mediated APB induction, which suggests that HP1-mediated chromatin compaction is required for APB formation. Therefore, although the presence of APBs in a cell line or tumor is an excellent qualitative marker for ALT, the association of APBs with growth arrest/senescence and with "closed" telomeric chromatin, which is likely to repress recombination, suggests there is no simple correlation between ALT activity level and the number of APBs or APB-positive cells.


Assuntos
Proteínas Cromossômicas não Histona/fisiologia , Telômero/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Antígenos Virais de Tumores/metabolismo , Linhagem Celular , Senescência Celular/genética , Senescência Celular/fisiologia , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Quinase 2 Dependente de Ciclina/análise , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Humanos , Proteínas Nucleares/análise , Proteínas Nucleares/metabolismo , Interferência de RNA , Proteína 1 de Ligação a Repetições Teloméricas/análise , Proteína 2 de Ligação a Repetições Teloméricas/análise , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima
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