Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 19 de 19
Filtrar
1.
Nature ; 629(8014): 1149-1157, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38720070

RESUMO

In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.


Assuntos
Cromatina , Epigênese Genética , Genótipo , Mutação , Análise de Célula Única , Animais , Feminino , Humanos , Masculino , Camundongos , Antígenos CD34/metabolismo , Diferenciação Celular/genética , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética/genética , Epigenoma/genética , Genoma Mitocondrial/genética , Técnicas de Genotipagem , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Inflamação/genética , Inflamação/patologia , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Megacariócitos/metabolismo , Megacariócitos/patologia , Proteínas de Membrana/genética , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Transtornos Mieloproliferativos/patologia , RNA/genética , Células Clonais/metabolismo
2.
Cancer Discov ; 14(5): 737-751, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38230747

RESUMO

Gain-of-function mutations activating JAK/STAT signaling are seen in the majority of patients with myeloproliferative neoplasms (MPN), most commonly JAK2V617F. Although clinically approved JAK inhibitors improve symptoms and outcomes in MPNs, remissions are rare, and mutant allele burden does not substantively change with chronic therapy. We hypothesized this is due to limitations of current JAK inhibitors to potently and specifically abrogate mutant JAK2 signaling. We therefore developed a conditionally inducible mouse model allowing for sequential activation, and then inactivation, of Jak2V617F from its endogenous locus using a combined Dre-rox/Cre-lox dual-recombinase system. Jak2V617F deletion abrogates MPN features, induces depletion of mutant-specific hematopoietic stem/progenitor cells, and extends overall survival to an extent not observed with pharmacologic JAK inhibition, including when cooccurring with somatic Tet2 loss. Our data suggest JAK2V617F represents the best therapeutic target in MPNs and demonstrate the therapeutic relevance of a dual-recombinase system to assess mutant-specific oncogenic dependencies in vivo. SIGNIFICANCE: Current JAK inhibitors to treat myeloproliferative neoplasms are ineffective at eradicating mutant cells. We developed an endogenously expressed Jak2V617F dual-recombinase knock-in/knock-out model to investigate Jak2V617F oncogenic reversion in vivo. Jak2V617F deletion abrogates MPN features and depletes disease-sustaining MPN stem cells, suggesting improved Jak2V617F targeting offers the potential for greater therapeutic efficacy. See related commentary by Celik and Challen, p. 701. This article is featured in Selected Articles from This Issue, p. 695.


Assuntos
Janus Quinase 2 , Transtornos Mieloproliferativos , Animais , Humanos , Camundongos , Modelos Animais de Doenças , Células-Tronco Hematopoéticas/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Mutação , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/tratamento farmacológico , Transdução de Sinais
3.
Blood Adv ; 8(2): 429-440, 2024 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-37871309

RESUMO

ABSTRACT: Enasidenib (ENA) is an inhibitor of isocitrate dehydrogenase 2 (IDH2) approved for the treatment of patients with IDH2-mutant relapsed/refractory acute myeloid leukemia (AML). In this phase 2/1b Beat AML substudy, we applied a risk-adapted approach to assess the efficacy of ENA monotherapy for patients aged ≥60 years with newly diagnosed IDH2-mutant AML in whom genomic profiling demonstrated that mutant IDH2 was in the dominant leukemic clone. Patients for whom ENA monotherapy did not induce a complete remission (CR) or CR with incomplete blood count recovery (CRi) enrolled in a phase 1b cohort with the addition of azacitidine. The phase 2 portion assessing the overall response to ENA alone demonstrated efficacy, with a composite complete response (cCR) rate (CR/CRi) of 46% in 60 evaluable patients. Seventeen patients subsequently transitioned to phase 1b combination therapy, with a cCR rate of 41% and 1 dose-limiting toxicity. Correlative studies highlight mechanisms of clonal elimination with differentiation therapy as well as therapeutic resistance. This study demonstrates both efficacy of ENA monotherapy in the upfront setting and feasibility and applicability of a risk-adapted approach to the upfront treatment of IDH2-mutant AML. This trial is registered at www.clinicaltrials.gov as #NCT03013998.


Assuntos
Aminopiridinas , Azacitidina , Leucemia Mieloide Aguda , Triazinas , Humanos , Azacitidina/efeitos adversos , Isocitrato Desidrogenase/genética , Mutação , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Resposta Patológica Completa
4.
Blood ; 141(20): 2508-2519, 2023 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-36800567

RESUMO

Proinflammatory signaling is a hallmark feature of human cancer, including in myeloproliferative neoplasms (MPNs), most notably myelofibrosis (MF). Dysregulated inflammatory signaling contributes to fibrotic progression in MF; however, the individual cytokine mediators elicited by malignant MPN cells to promote collagen-producing fibrosis and disease evolution are yet to be fully elucidated. Previously, we identified a critical role for combined constitutive JAK/STAT and aberrant NF-κB proinflammatory signaling in MF development. Using single-cell transcriptional and cytokine-secretion studies of primary cells from patients with MF and the human MPLW515L (hMPLW515L) murine model of MF, we extend our previous work and delineate the role of CXCL8/CXCR2 signaling in MF pathogenesis and bone marrow fibrosis progression. Hematopoietic stem/progenitor cells from patients with MF are enriched for a CXCL8/CXCR2 gene signature and display enhanced proliferation and fitness in response to an exogenous CXCL8 ligand in vitro. Genetic deletion of Cxcr2 in the hMPLW515L-adoptive transfer model abrogates fibrosis and extends overall survival, and pharmacologic inhibition of the CXCR1/2 pathway improves hematologic parameters, attenuates bone marrow fibrosis, and synergizes with JAK inhibitor therapy. Our mechanistic insights provide a rationale for therapeutic targeting of the CXCL8/CXCR2 pathway among patients with MF.


Assuntos
Transtornos Mieloproliferativos , Neoplasias , Mielofibrose Primária , Humanos , Camundongos , Animais , Mielofibrose Primária/patologia , Transtornos Mieloproliferativos/genética , Transdução de Sinais , Neoplasias/complicações , Citocinas/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/metabolismo
5.
Blood ; 136(1): 61-70, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32430500

RESUMO

Secondary acute myeloid leukemias (AMLs) evolving from an antecedent myeloproliferative neoplasm (MPN) are characterized by a unique set of cytogenetic and molecular features distinct from de novo AML. Given the high frequency of poor-risk cytogenetic and molecular features, malignant clones are frequently insensitive to traditional AML chemotherapeutic agents. Allogeneic stem cell transplant, the only treatment modality shown to have any beneficial long-term outcome, is often not possible given the advanced age of patients at time of diagnosis and frequent presence of competing comorbidities. Even in this setting, relapse rates remain high. As a result, outcomes are generally poor and there remains a significant unmet need for novel therapeutic strategies. Although advances in cancer genomics have dramatically enhanced our understanding of the molecular events governing clonal evolution in MPNs, the cell-intrinsic and -extrinsic mechanisms driving leukemic transformation at this level remain poorly understood. Here, we review known risk factors for the development of leukemic transformation in MPNs, recent progress made in our understanding of the molecular features associated with leukemic transformation, current treatment strategies, and emerging therapeutic options for this high-risk myeloid malignancy.


Assuntos
Leucemia Mieloide Aguda/etiologia , Transtornos Mieloproliferativos/patologia , Cariótipo Anormal , Aloenxertos , Antineoplásicos/uso terapêutico , Transformação Celular Neoplásica , Aberrações Cromossômicas , Evolução Clonal , Terapia Combinada , Comorbidade , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Drogas em Investigação/uso terapêutico , Genes Neoplásicos , Transplante de Células-Tronco Hematopoéticas , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/terapia , Modelos Biológicos , Mutação , Transtornos Mieloproliferativos/genética , Proteínas de Neoplasias/genética , Recidiva , Fatores de Risco , Análise de Célula Única , Terapias em Estudo
6.
Case Rep Hematol ; 2020: 2795656, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32148977

RESUMO

BACKGROUND: The identification of germline mutations in familial leukemia predisposition genes by next generation sequencing is of pivotal importance. Lately, some "blend pedigrees" characterized by both solid and hematologic malignancies have been described. Some genes were recognized as related to this double predisposition, while the involvement of others is still a matter of debate. ETV6 was associated with hematologic malignancies, in particular myeloid malignancies, and recently described as mutated also in oncologic patients. No clear evidences in its involvement in blend pedigrees are known. Case Presentation. We present our recent experience in the identification of an ETV6 was associated with hematologic malignancies, in particular myeloid malignancies, and recently described as mutated also in oncologic patients. No clear evidences in its involvement in blend pedigrees are known. ETV6 was associated with hematologic malignancies, in particular myeloid malignancies, and recently described as mutated also in oncologic patients. No clear evidences in its involvement in blend pedigrees are known. ETV6 was associated with hematologic malignancies, in particular myeloid malignancies, and recently described as mutated also in oncologic patients. No clear evidences in its involvement in blend pedigrees are known. CONCLUSION: This evidence supports the involvement of ETV6 in the predisposition to both solid and hematologic neoplasia and the importance of the investigation of the noncoding regions of the genes as recently suggested by different expert groups.ETV6 was associated with hematologic malignancies, in particular myeloid malignancies, and recently described as mutated also in oncologic patients. No clear evidences in its involvement in blend pedigrees are known.

7.
Haematologica ; 104(7): 1378-1387, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30523054

RESUMO

Measurable residual disease is associated with inferior outcomes in patients with acute myeloid leukemia (AML). Measurable residual disease monitoring enhances risk stratification and may guide therapeutic intervention. The European LeukemiaNet working party recently came to a consensus recommendation incorporating leukemia associated immunophenotype-based different from normal approach by multi-color flow cytometry for measurable residual disease evaluation. However, the analytical approach is highly expertise-dependent and difficult to standardize. Here we demonstrate that loss of plasmacytoid dendritic cell differentiation after 7+3 induction in AML is highly specific for measurable residual disease positivity (specificity 97.4%) in a uniformly treated patient cohort. Moreover, loss of plasmacytoid dendritic cell differentiation as determined by a blast-to-plasmacytoid dendritic cell ratio >10 was strongly associated with inferior overall and relapse-free survival (RFS) [Hazard ratio 2.79, 95% confidence interval (95%CI): 0.98-7.97; P=0.077) and 3.83 (95%CI: 1.51-9.74; P=0.007), respectively), which is similar in magnitude to measurable residual disease positivity. Importantly, measurable residual disease positive patients who reconstituted plasmacytoid dendritic cell differentiation (blast/ plasmacytoid dendritic cell ratio <10) showed a higher rate of measurable residual disease clearance at later pre-transplant time points compared to patients with loss of plasmacytoid dendritic cell differentiation (blast/ plasmacytoid dendritic cell ratio <10) (6 of 12, 50% vs 2 of 18, 11%; P=0.03). Furthermore pre-transplant plasmacytoid dendritic cell recovery was associated with superior outcome in measurable residual disease positive patients. Our study provides a novel, simple, broadly applicable, and quantitative multi-color flow cytometry approach to risk stratification in AML.


Assuntos
Células Dendríticas/patologia , Leucemia Mieloide Aguda/mortalidade , Recidiva Local de Neoplasia/mortalidade , Neoplasia Residual/mortalidade , Adulto , Idoso , Estudos de Casos e Controles , Terapia Combinada , Feminino , Seguimentos , Humanos , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/terapia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/terapia , Neoplasia Residual/patologia , Neoplasia Residual/terapia , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida
8.
Cancer Res ; 68(24): 10349-57, 2008 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19074904

RESUMO

Two types of acquired loss of heterozygosity are possible in cancer: deletions and copy-neutral uniparental disomy (UPD). Conventionally, copy number losses are identified using metaphase cytogenetics, whereas detection of UPD is accomplished by microsatellite and copy number analysis and as such, is not often used clinically. Recently, introduction of single nucleotide polymorphism (SNP) microarrays has allowed for the systematic and sensitive detection of UPD in hematologic malignancies and other cancers. In this study, we have applied 250K SNP array technology to detect previously cryptic chromosomal changes, particularly UPD, in a cohort of 301 patients with myelodysplastic syndromes (MDS), overlap MDS/myeloproliferative disorders (MPD), MPD, and acute myeloid leukemia. We show that UPD is a common chromosomal defect in myeloid malignancies, particularly in chronic myelomonocytic leukemia (CMML; 48%) and MDS/MPD-unclassifiable (38%). Furthermore, we show that mapping minimally overlapping segmental UPD regions can help target the search for both known and unknown pathogenic mutations, including newly identified missense mutations in the proto-oncogene c-Cbl in 7 of 12 patients with UPD11q. Acquired mutations of c-Cbl E3 ubiquitin ligase may explain the pathogenesis of a clonal process in a subset of MDS/MPD, including CMML.


Assuntos
Mutação , Doenças Mieloproliferativas-Mielodisplásicas/genética , Proteínas Proto-Oncogênicas c-cbl/genética , Dissomia Uniparental/genética , Adolescente , Adulto , Idoso , Humanos , Cariotipagem , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Mutação Puntual , Polimorfismo de Nucleotídeo Único , Proto-Oncogene Mas , Adulto Jovem
9.
PLoS One ; 2(11): e1225, 2007 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-18030353

RESUMO

We applied single nucleotide polymorphism arrays (SNP-A) to study karyotypic abnormalities in patients with atypical myeloproliferative syndromes (MPD), including myeloproliferative/myelodysplastic syndrome overlap both positive and negative for the JAK2 V617F mutation and secondary acute myeloid leukemia (AML). In typical MPD cases (N = 8), which served as a control group, those with a homozygous V617F mutation showed clear uniparental disomy (UPD) of 9p using SNP-A. Consistent with possible genomic instability, in 19/30 MDS/MPD-U patients, we found additional lesions not identified by metaphase cytogenetics. In addition to UPD9p, we also have detected UPD affecting other chromosomes, including 1 (2/30), 11 (4/30), 12 (1/30) and 22 (1/30). Transformation to AML was observed in 8/30 patients. In 5 V617F+ patients who progressed to AML, we show that SNP-A can allow for the detection of two modes of transformation: leukemic blasts evolving from either a wild-type jak2 precursor carrying other acquired chromosomal defects, or from a V617F+ mutant progenitor characterized by UPD9p. SNP-A-based detection of cryptic lesions in MDS/MPD-U may help explain the clinical heterogeneity of this disorder.


Assuntos
Aberrações Cromossômicas , Cariotipagem , Síndromes Mielodisplásicas/genética , Transtornos Mieloproliferativos/genética , Polimorfismo de Nucleotídeo Único , Dissomia Uniparental , Sequência de Bases , Primers do DNA , Humanos , Reação em Cadeia da Polimerase
10.
Semin Thromb Hemost ; 33(4): 397-407, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17525897

RESUMO

Tumor lysis syndrome (TLS) is an important metabolic disorder frequently encountered in the management of a variety of cancers including lymphoma, leukemia, and neuroblastoma. Delayed recognition can result in a variety of biochemical abnormalities resulting in life-threatening complications such as renal failure, arrhythmias, and seizures. Identification of high-risk patients and early recognition of the syndrome is crucial in the early institution of appropriate prophylaxis and treatment. Recent advances in the understanding of urate metabolism, development of new urate-lowering drugs, and the application of biomarkers, calculation methods, and prognostic models to identify high-risk patients will pave the way in improving the management of TLS. We included in this review the new information regarding the urate transporters URAT-1, organic anion transporter 1/3, and MRP4; the urate elimination pathway; a comparison of the old- (allopurinol, native uricase) and new- (febuxostat, Y-700, PEG-uricase, rasburicase) generation urate-lowering agents; and application of new biomarkers (cystatin-C, neutrophil gelatinase-associated lipocalin, kidney injury molecule 1), estimated glomerular filtration rate and calculation methods (modification of diet in renal disease and prognostic model (Penn Predictive Score of Tumor Lysis Syndrome) in the identification of high-risk patients, and alternative unexplored mechanisms (asymmetric dimethylarginine and adenosine) to explain renal injury related to TLS.


Assuntos
Síndrome de Lise Tumoral/diagnóstico , Biomarcadores/análise , Humanos , Nefropatias/etiologia , Neoplasias/complicações , Ácido Úrico/metabolismo
11.
Growth Factors ; 24(2): 121-36, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16801132

RESUMO

Epidermal growth factor (EGF)-like proteins comprise a group of structurally similar growth factors, which contain a conserved six-cysteine residue motif called the EGF-domain. EGF-like factors are synthesized as transmembrane precursors, which can undergo proteolytic cleavage at the cell surface to release a mature soluble ectodomain; a process often referred to as "ectodomain shedding". Ectodomain shedding of EGF-like factors has been linked to multiple zinc-binding metalloproteases of the matrix metalloprotease (MMP) and a disintegrin and metalloprotease (ADAM) families. Shedding can be activated by a variety of pharmacological and physiological stimuli and these activation events have been linked to the enhancement of metalloprotease activity, possibly via the action of intracellular signaling modules. Once shed from the cell surface, EGF-like factors bind to a family of four cell surface receptors named ErbB-1, -2, -3 and -4. Heterodimerization or homodimerization of these receptors following ligand binding drives intracellular signal transduction cascades, which eventuate in diverse cell fates including proliferation, differentiation, migration and inhibition of apoptosis. In addition to its role in driving normal developmental processes, a wealth of evidence now exists showing that de-regulated ErbB signaling is associated with the formation of tumors in a variety of tissues and that ectodomain shedding of EGF-like factors plays a critical event in this process. Thus, knowledge of the molecular mechanisms by which EGF-like factors are shed from the cell surface and the nature of the proteases and cellular signals that govern this process is crucial to understanding ErbB receptor signaling and potentially also in the development of novel cancer therapeutics targeting the ErbB pathway. This review focuses on the structure and function of EGF-like factors, and the mechanisms that govern the shedding of these transmembrane molecules from the cell surface.


Assuntos
Fator de Crescimento Epidérmico/fisiologia , Receptores ErbB/metabolismo , Metaloproteases/fisiologia , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Sequência de Aminoácidos , Animais , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/metabolismo , Crescimento e Desenvolvimento/fisiologia , Humanos , Metaloproteases/química , Dados de Sequência Molecular , Neoplasias/fisiopatologia , Estrutura Terciária de Proteína , Transdução de Sinais/fisiologia
12.
J Cell Biochem ; 99(2): 609-23, 2006 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-16676357

RESUMO

The betacellulin precursor (pro-BTC) is a novel substrate for ADAM10-mediated ectodomain shedding. In this report, we investigated the ability of novel physiologically relevant stimuli, including G-protein coupled receptor (GPCR) agonists and reactive oxygen species (ROS), to stimulate pro-BTC shedding. We found that in breast adenocarcinoma MCF7 cells overexpressing pro-BTC, hydrogen peroxide (H2O2) was a powerful stimulator of ectodomain shedding. The stimulation of pro-BTC shedding by H2O2 was blocked by the broad-spectrum metalloprotease inhibitor TAPI-0 but was still functional in ADAM17 (TACE)-deficient stomach epithelial cells indicating the involvement of a distinct metalloprotease. H2O2-induced pro-BTC shedding was blocked by co-culturing cells in the anti-oxidant N-acetyl-L-cysteine but was unaffected by culture in calcium-deficient media. By contrast, calcium ionophore, which is a previously characterized activator of pro-BTC shedding, was sensitive to calcium depletion but was unaffected by co-culture with the anti-oxidant, identifying a clear distinction between these stimuli. We found that in vascular smooth muscle cells overexpressing pro-BTC, the GPCR agonist endothelin-1 (ET-1) was a strong inducer of ectodomain shedding. This was blocked by a metalloprotease inhibitor and by overexpression of catalytically inactive E385A ADAM10. However, overexpression of wild-type ADAM10 or ADAM17 led to an increase in ET-1-induced pro-BTC shedding providing evidence for an involvement of both enzymes in this process. This study identifies ROS and ET-1 as two novel inducers of pro-BTC shedding and lends support to the notion of activated shedding occurring under the control of physiologically relevant stimuli.


Assuntos
Endotelina-1/farmacologia , Peróxido de Hidrogênio/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM10 , Proteína ADAM17 , Secretases da Proteína Precursora do Amiloide , Animais , Betacelulina , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Calcimicina/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Dipeptídeos/farmacologia , Endotelina-1/metabolismo , Feminino , Humanos , Ácidos Hidroxâmicos/farmacologia , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metaloendopeptidases/antagonistas & inibidores , Camundongos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Estrutura Terciária de Proteína , Coelhos , Receptores Acoplados a Proteínas G/agonistas
13.
Dev Biol ; 287(1): 146-56, 2005 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16188250

RESUMO

Ammonium transporter C (AmtC) is one of three transporters in Dictyostelium that have been proposed to regulate entry and exit of ammonia in a cell type dependent manner and to mediate ammonia signaling. Previous work demonstrated that disruption of the amtC gene results in a slugger phenotype in which the cells remain as migrating slugs when they should form fruiting bodies. More detailed studies on the null strain revealed that differentiation of prestalk cell types was delayed and maintenance of prestalk cell gene expression was defective. There was little or no expression of ecmB, a marker for the initiation of culmination. Normal expression of CudA, a nuclear protein required for culmination, was absent in the anterior prestalk zone. The absence of CudA within the tip region was attributable to the lack of nuclear localization of the transcription factor STATa, despite expression of adenylyl cyclase A mRNA in the slug tips. Disruption of the histidine kinase gene dhkC in the amtC null strain restored STATa and CudA expression and the ability to culminate. The results suggest that the lack of nuclear translocation of STATa results from low cAMP due to a misregulated and overactive DhkC phosphorelay in the amtC null strain.


Assuntos
Proteínas de Transporte de Cátions/fisiologia , Movimento Celular/fisiologia , Dictyostelium/fisiologia , Regulação da Expressão Gênica/fisiologia , Compostos de Amônio Quaternário/metabolismo , Animais , Proteínas de Transporte de Cátions/genética , Núcleo Celular/metabolismo , Dictyostelium/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Proteínas Quinases/biossíntese , Proteínas Quinases/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Fatores de Transcrição STAT/metabolismo , Regulação para Cima
14.
Endocrinology ; 146(11): 4673-81, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16081630

RESUMO

We previously described a novel alternatively spliced mRNA transcript of the betacellulin (BTC) gene. This splice isoform, termed BTC-delta4, lacks the C-loop of the epidermal growth factor motif and the transmembrane domain as a result of exon 4 'skipping'. In this study, we expressed BTC-delta4 recombinantly to explore its biological function. When BTC-delta4 was expressed in COS-7 cells, it was secreted largely into the culture medium, in contrast to BTC. Unlike BTC, highly purified recombinant BTC-delta4 produced in Escherichia coli failed to bind or induce tyrosine phosphorylation of either ErbB1 or ErbB4, nor did it antagonize the binding of BTC to these receptors. Consistent with this, BTC-delta4 failed to stimulate DNA synthesis in Balb/c 3T3 and INS-1 cells. However, BTC-delta4 induced differentiation of pancreatic beta-cells; BTC-delta4 converted AR42J cells to insulin-producing cells. When recombinant BTC-delta4 was administered to streptozotocin-treated neonatal rats, it reduced the plasma glucose concentration and improved glucose tolerance. Importantly, BTC-delta4 significantly increased the insulin content, the beta-cell mass, and the numbers of islet-like cell clusters and PDX-1-positive ductal cells. Thus, BTC-delta4 is a secreted protein that stimulates differentiation of beta-cells in vitro and in vivo in an apparent ErbB1- and ErbB4-independent manner. The mechanism by which BTC-delta4 exerts this action on beta-cells remains to be defined but presumably involves an, as yet, unidentified unique receptor.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Intolerância à Glucose/induzido quimicamente , Intolerância à Glucose/fisiopatologia , Células Secretoras de Insulina/patologia , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Estreptozocina , Animais , Betacelulina , Glicemia/metabolismo , Linhagem Celular , Receptores ErbB/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Concentração Osmolar , Ratos , Ratos Sprague-Dawley , Receptor ErbB-2/metabolismo , Receptor ErbB-4 , Proteínas Recombinantes/biossíntese
15.
J Biol Chem ; 280(3): 1826-37, 2005 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-15507448

RESUMO

Betacellulin belongs to the family of epidermal growth factor-like growth factors that are expressed as transmembrane precursors and undergo proteolytic ectodomain shedding to release a soluble mature growth factor. In this study, we investigated the ectodomain shedding of the betacellulin precursor (pro-BTC) in conditionally immortalized wild-type (WT) and ADAM-deficient cell lines. Sequential ectodomain cleavage of the predominant cell-surface 40-kDa form of pro-BTC generated a major (26-28 kDa) and two minor (20 and 15 kDa) soluble forms and a cellular remnant lacking the ectodomain (12 kDa). Pro-BTC shedding was activated by calcium ionophore (A23187) and by the metalloprotease activator p-aminophenylmercuric acetate (APMA), but not by phorbol esters. Culturing cells in calcium-free medium or with the protein kinase Cdelta inhibitor rottlerin, but not with broad-based protein kinase C inhibitors, blocked A23187-activated pro-BTC shedding. These same treatments were without effect for constitutive and APMA-induced cleavage events. All pro-BTC shedding was blocked by treatment with a broad-spectrum metalloprotease inhibitor (GM6001). In addition, constitutive and activated pro-BTC shedding was differentially blocked by TIMP-1 or TIMP-3, but was insensitive to treatment with TIMP-2. Pro-BTC shedding was functional in cells from ADAM17- and ADAM9-deficient mice and in cells overexpressing WT or catalytically inactive ADAM17. In contrast, overexpression of WT ADAM10 enhanced constitutive and activated shedding of pro-BTC, whereas overexpression of catalytically inactive ADAM10 reduced shedding. These results demonstrate, for the first time, activated pro-BTC shedding in response to extracellular calcium influx and APMA and provide evidence that ADAM10 mediates constitutive and activated pro-BTC shedding.


Assuntos
Cálcio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/fisiologia , Metaloendopeptidases/fisiologia , Acetato de Fenilmercúrio/análogos & derivados , Acetato de Fenilmercúrio/farmacologia , Proteínas ADAM , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide , Sequência de Bases , Betacelulina , Linhagem Celular Transformada , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Humanos , Transporte de Íons
16.
Growth Factors ; 21(2): 79-86, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-14626355

RESUMO

Betacellulin is a relatively new member of the epidermal growth factor peptide family, however, its function remains poorly defined. We investigated its physiological effects in rats implanted with pumps to deliver vehicle or recombinant rat betacellulin [46 microg/day] for 7 days. At kill, blood and gastrointestinal tissues were collected for determinations of betacellulin levels, proliferation (bromodeoxyuridine-BrdU incorporation) and growth. Plasma betacellulin levels were increased 8-fold compared to vehicle, whilst serum insulin, body weight and food intake were decreased by 32, 15 and 9%, respectively. Water intake, urine and faecal output and small intestinal weight were respectively increased by 36, 78, 47 and 24%. Ileal and proximal colonic crypt depths were increased by 25 and 51% although the BrdU labelling index was unaffected. Betacellulin stimulated gastrointestinal growth, the increased responsiveness of the terminal ileum and colon suggesting therapeutic potential in disease conditions in which ileal or colonic re-growth is desirable. Betacellulin further stimulated a diuresis suggesting an additional role in fluid homeostasis.


Assuntos
Sistema Digestório/crescimento & desenvolvimento , Diurese/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Animais , Betacelulina , Glicemia/análise , Sistema Digestório/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Masculino , Tamanho do Órgão/efeitos dos fármacos , Espaço Pessoal , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Fatores de Tempo
17.
Biochim Biophys Acta ; 1576(1-2): 183-90, 2002 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-12031500

RESUMO

Betacellulin (BTC) belongs to the epidermal growth factor (EGF) family of peptide ligands that are characterized by a six-cysteine consensus motif (EGF-motif) that forms three intra-molecular disulfide bonds, crucial for binding the ErbB receptor family. A variety of in vitro studies have identified BTC as an important factor in the growth and/or differentiation of pancreatic islet cells. The molecular mechanisms that regulate the transcription of the BTC gene however have not been delineated. As an initial step, we have characterized the genomic structure of the mouse BTC (mBTC) gene. mBTC cDNA was used as a probe to screen a mouse 129/SVJ genomic bacterial artificial chromosome (BAC) library. Three positive clones containing the entire gene were isolated. DNA sequence analysis identified six exons (1-6) and five introns (A-E); a structure conserved among the EGF family. PCR analysis showed that introns A-E are approximately 7.8, 8.9, 3.8, 1.4 and 1.4 kb in length, respectively. The EGF-motif is encoded by exons 3 and 4 with an intron (intron C) disrupting the coding sequence between the second and third disulfide loops. All exon-intron boundaries are consistent with the "gt-ag" rule. Multiple transcription start sites and one poly(A) site, located 18 bp downstream of a polyadenylation signal sequence, were identified by 5'- and 3'-RACE, respectively. Approximately 2.6 kb of 5'-flanking region was sequenced and was shown to lack consensus TATA and CCAAT boxes, but was found to contain several putative cis-acting regulatory elements. These included consensus binding sites for transcription factors HNF3 beta, USF, Nkx2-5, AP-4, and Sp1. Functional promoter analysis of the 5'-flanking region in COS-7 cells, using 5'-deletion fragments (-168/+335; -635/+335; -732/+335; -1175/+335; -1698/+335) cloned into a promoterless firefly luciferase reporter vector, identified basal promoter activity and both positive and negative cis-acting elements.


Assuntos
Fator de Crescimento Epidérmico/genética , Substâncias de Crescimento/genética , Peptídeos e Proteínas de Sinalização Intercelular , Regiões 5' não Traduzidas/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Betacelulina , Sítios de Ligação , Sequência Consenso , Éxons , Deleção de Genes , Biblioteca Genômica , Íntrons , Camundongos , Dados de Sequência Molecular , Pâncreas/metabolismo , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas/genética
18.
Microbiology (Reading) ; 145 ( Pt 8): 1891-1901, 1999 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10463155

RESUMO

The sori of Dictyostelium discoideum (strains SG1, SG2, NC4 and V12) contained more than 100 mM ammonium phosphate. Glutamine synthetase (GS), which could remove ammonia from the sorus, was not present in 2-d-old dormant spores but enzyme activity returned to vegetative levels after spore germination. Based on mRNA blotting, the activity of this enzyme in germinating spores appeared to be transcriptionally controlled. At the same time that GS activity was increasing, ammonia was released from germinating spores. Exogenous ammonium ions at a concentration of 28 mM did not block germination nor modulate GS activity in nascent amoebae. It was concluded that the transcription and translation of GS is not environmentally regulated but is an integral part of the germination process, preparing nascent amoebae for vegetative growth. An exogenous concentration of 69 mM ammonium phosphate could maintain dormancy in spores of strains SG1 and SG2 for at least a week in the absence of any other inhibitory component from the sori. The inhibition was reversible at any time either by dilution or by washing the spores free of the ammonium ion. Spores of strain acg- were not inhibited by 100 mM ammonium phosphate. A model is presented in which GS in prespore cells serves as a sink for ammonia to allow the osmotically sensitive adenylyl cyclase aggregation protein (ACA) to activate protein kinase A (PKA) to induce fruiting-body formation. After fruiting-body formation is complete, the decline in GS and ACA activities in developing spores is offset by their replacement with the osmotically and ammonia-stimulated adenylyl cyclase osmosensor for germination (ACG). Ammonia and discadenine may act as separate signals to synergistically activate PKA by stimulating ACG activity while inhibiting cAMP phosphodiestrase activity in fully dormant spores.


Assuntos
Adenilil Ciclases/metabolismo , Dictyostelium/fisiologia , Fosfatos/metabolismo , Proteínas de Protozoários , Amônia/metabolismo , Animais , Northern Blotting , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica no Desenvolvimento , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/metabolismo , Fosfatos/farmacologia , Esporos/fisiologia , Transcrição Gênica
19.
Microbiology (Reading) ; 141 ( Pt 5): 1125-1130, 1995 May.
Artigo em Inglês | MEDLINE | ID: mdl-7773407

RESUMO

The enzyme glutamine synthetase (GS) is described for the first time in Dictyostelium discoideum. The appearance of this enzyme is developmentally regulated. The level of activity is low in vegetative cells and increases more than threefold during differentiation. Furthermore this enzyme is shown to be differentially localized in prespore cells, the specific activity being approximately fourfold higher than in prestalk cells. The enzyme has a pH optimum of 7.8 and 8.2 in the gamma-glutamyltransferase and gamma-glutamylsynthetase assays, respectively, and a temperature optimum of 45 degrees C. Kinetic studies of GS revealed apparent Km values of 5.9 mM, 0.009 mM and 8.6 mM for glutamine, ADP and NH2OH, respectively, in the gamma-glutamyltransferase assay, and of 2.2 mM, 0.12 mM and 0.64 mM for glutamate, ATP and NH2OH, respectively, in the gamma-glutamylsynthetase assay.


Assuntos
Dictyostelium/fisiologia , Glutamato-Amônia Ligase/metabolismo , Animais , Dictyostelium/enzimologia , Regulação Fúngica da Expressão Gênica , Glutamato-Amônia Ligase/biossíntese , Cinética , Esporos Fúngicos , Fatores de Tempo , gama-Glutamiltransferase/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...