RESUMO
PURPOSE: We investigated the translational value of reflex testing for germline mutations in four homology-directed DNA repair predisposition genes (BRCA1, BRCA2, PALB2, and ATM) in consecutive patients with pancreatic adenocarcinoma. METHODS: One hundred fifty patients with French-Canadian (FC) ancestry were evaluated for founder mutations, and 114 patients were subsequently assessed by full gene sequencing and multiplex ligation-dependent probe amplification for nonfounder mutations. Two hundred thirty-six patients unselected for ancestry were also assessed for mutations by full gene sequencing. RESULTS: The FC founder mutation prevalence among the 150 patients was 5.3% (95% CI, 2.6% to 10.3%), and the nonfounder mutation prevalence across the four genes among the 114 patients tested was 2.6% (95% CI, 0.6% to 7.8%). In the case series unselected for ancestry, 10.0% (95% CI, 2.7% to 26.4%) of patients reporting Ashkenazi Jewish (AJ) ancestry carried an AJ founder mutation, with no nonfounder mutations identified. The mutation prevalence among patients without FC/AJ ancestry was 4.9% (95% CI, 2.6% to 8.8%). Mutations were more frequent in patients diagnosed at ≤ 50 years of age (P = .03) and in patients with either two or more first- or second-degree relatives with pancreas, breast, ovarian or prostate cancer, or one such relative and a second primary of one of these cancer types (P < .001). BRCA1, BRCA2, and PALB2 carriers with late-stage (III or IV) disease had an overall survival advantage (P = .049), particularly if treated with platinum-based chemotherapies (P = .030). CONCLUSION: Considering these results, we recommend reflex founder mutation testing of patients with FC/AJ ancestry and full gene sequencing of patients who are ≤ 50 years or meet the identified family history criteria. Reflex testing of all incident patients for these four genes may become justified as full gene sequencing costs decline.
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PTP1B and TC-PTP are highly related protein-tyrosine phosphatases (PTPs) that regulate the JAK/STAT signaling cascade essential for cytokine-receptor activation in immune cells. Here, we describe a novel immunotherapy approach whereby monocyte-derived dendritic cell (moDC) function is enhanced by modulating the enzymatic activities of PTP1B and TC-PTP. To downregulate or delete the activity/expression of these PTPs, we generated mice with PTP-specific deletions in the dendritic cell compartment or used PTP1B and TC-PTP specific inhibitor. While total ablation of PTP1B or TC-PTP expression leads to tolerogenic DCs via STAT3 hyperactivation, downregulation of either phosphatase remarkably shifts the balance toward an immunogenic DC phenotype due to hyperactivation of STAT4, STAT1 and Src kinase. The resulting increase in IL-12 and IFNγ production subsequently amplifies the IL-12/STAT4/IFNγ/STAT1/IL-12 positive autocrine loop and enhances the therapeutic potential of mature moDCs in tumor-bearing mice. Furthermore, pharmacological inhibition of both PTPs improves the maturation of defective moDCs derived from pancreatic cancer (PaC) patients. Our study provides a new advance in the use of DC-based cancer immunotherapy that is complementary to current cancer therapeutics.
RESUMO
Microtubule-targeting agents (MTAs) are widely used anticancer agents, but toxicities such as neuropathy limit their clinical use. MTAs bind to and alter the stability of microtubules, causing cell death in mitosis. We describe DZ-2384, a preclinical compound that exhibits potent antitumor activity in models of multiple cancer types. It has an unusually high safety margin and lacks neurotoxicity in rats at effective plasma concentrations. DZ-2384 binds the vinca domain of tubulin in a distinct way, imparting structurally and functionally different effects on microtubule dynamics compared to other vinca-binding compounds. X-ray crystallography and electron microscopy studies demonstrate that DZ-2384 causes straightening of curved protofilaments, an effect proposed to favor polymerization of tubulin. Both DZ-2384 and the vinca alkaloid vinorelbine inhibit microtubule growth rate; however, DZ-2384 increases the rescue frequency and preserves the microtubule network in nonmitotic cells and in primary neurons. This differential modulation of tubulin results in a potent MTA therapeutic with enhanced safety.
Assuntos
Antineoplásicos/farmacologia , Lactamas Macrocíclicas/farmacologia , Microtúbulos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Oxazóis/farmacologia , Alcaloides de Vinca/farmacologia , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Cristalografia por Raios X , Dimerização , Genômica , Humanos , Lactamas Macrocíclicas/química , Camundongos , Microscopia Eletrônica , Mitose , Transplante de Neoplasias , Oxazóis/química , Tubulina (Proteína)/química , Vimblastina/análogos & derivados , Vimblastina/química , Vimblastina/farmacologia , Alcaloides de Vinca/química , VinorelbinaRESUMO
BACKGROUND & AIMS: Incidence of and mortality from pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, are almost equivalent, so better treatments are needed. We studied gene expression profiles of PDACs and the functions of genes with altered expression to identify new therapeutic targets. METHODS: We performed microarray analysis to analyze gene expression profiles of 195 PDAC and 41 non-tumor pancreatic tissue samples. We undertook an extensive analysis of the PDAC transcriptome by superimposing interaction networks of proteins encoded by aberrantly expressed genes over signaling pathways associated with PDAC development to identify factors that might alter regulation of these pathways during tumor progression. We performed tissue microarray analysis to verify changes in expression of candidate protein using an independent set of 152 samples (40 nontumor pancreatic tissues, 63 PDAC sections, and 49 chronic pancreatitis samples). We validated the functional relevance of the candidate molecule using RNA interference or pharmacologic inhibitors in pancreatic cancer cell lines and analyses of xenograft tumors in mice. RESULTS: In an analysis of 38,276 human genes and loci, we identified 1676 genes that were significantly up-regulated and 1166 genes that were significantly down-regulated in PDAC compared with nontumor pancreatic tissues. One gene that was up-regulated and associated with multiple signaling pathways that are dysregulated in PDAC was G protein subunit αi2, which has not been previously associated with PDAC. G protein subunit αi2 mediates the effects of dopamine receptor D2 (DRD2) on cyclic adenosine monophosphate signaling; PDAC tissues had a slight but significant increase in DRD2 messenger RNA. Levels of DRD2 protein were substantially increased in PDACs, compared with non-tumor tissues, in tissue microarray analyses. RNA interference knockdown of DRD2 or inhibition with pharmacologic antagonists (pimozide and haloperidol) reduced proliferation of pancreatic cancer cells, induced endoplasmic reticulum stress and apoptosis, and reduced cell migration. RNA interference knockdown of DRD2 in pancreatic tumor cells reduced growth of xenograft tumors in mice, and administration of the DRD2 inhibitor haloperidol to mice with orthotopic xenograft tumors reduced final tumor size and metastasis. CONCLUSIONS: In gene expression profile analysis of PDAC samples, we found the DRD2 signaling pathway to be activated. Inhibition of DRD2 in pancreatic cancer cells reduced proliferation and migration, and slowed growth of xenograft tumors in mice. DRD2 antagonists routinely used for management of schizophrenia might be tested in patients with pancreatic cancer.
Assuntos
Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Receptores de Dopamina D2/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/efeitos dos fármacos , Carcinoma Ductal Pancreático/secundário , Estudos de Casos e Controles , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/genética , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Antagonistas dos Receptores de Dopamina D2/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Haloperidol/farmacologia , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Neoplasias Pancreáticas/patologia , Fosforilação/efeitos dos fármacos , Pimozida/farmacologia , RNA Interferente Pequeno , Receptores de Dopamina D2/metabolismo , Transdução de Sinais , Transcriptoma , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Regulação para Cima , eIF-2 Quinase/metabolismoRESUMO
The genetic basis underlying the majority of hereditary pancreatic adenocarcinoma (PC) is unknown. Since DNA repair genes are widely implicated in gastrointestinal malignancies, including PC, we hypothesized that there are novel DNA repair PC susceptibility genes. As germline DNA repair gene mutations may lead to PC subtypes with selective therapeutic responses, we also hypothesized that there is an overall survival (OS) difference in mutation carriers versus non-carriers. We therefore interrogated the germline exomes of 109 high-risk PC cases for rare protein-truncating variants (PTVs) in 513 putative DNA repair genes. We identified PTVs in 41 novel genes among 36 kindred. Additional genetic evidence for causality was obtained for 17 genes, with FAN1, NEK1 and RHNO1 emerging as the strongest candidates. An OS difference was observed for carriers versus non-carriers of PTVs with early stage (≤IIB) disease. This adverse survival trend in carriers with early stage disease was also observed in an independent series of 130 PC cases. We identified candidate DNA repair PC susceptibility genes and suggest that carriers of a germline PTV in a DNA repair gene with early stage disease have worse survival.
Assuntos
Reparo do DNA/genética , Exoma , Neoplasias Pancreáticas/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Humanos , Quinase 1 Relacionada a NIMA , Neoplasias Pancreáticas/mortalidade , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Fatores de Risco , Análise de Sequência de DNARESUMO
BRCA2-associated pancreatic ductal adenocarcinoma (PDAC) may be sensitive to agents that target homology-directed DNA repair, such as DNA crosslinking agents (DCLs) and PARP inhibitors (PARPis). Here, we assessed the sensitivities of BRCA2-deficient (Capan-1) and BRCA2-proficient (MIA PaCa-2) PDAC cell lines to a panel of DCLs and PARPis. Compared to MIA PaCa-2, Capan-1 was significantly more sensitive to all tested DCLs and PARPis, with similar increased sensitivities to cisplatin and the PARPi BMN 673 compared to other DCLs and the PARPi veliparib. We provide further support for this observation by showing that shRNA-mediated BRCA2 knockdown in PANC-1, a BRCA2-proficient cell line, induces sensitization to cisplatin and BMN 673 but not to veliparib. These findings were validated in a PDAC murine xenograft model derived from a patient with bi-allelic BRCA2 mutations. We found 64% and 61% tumor growth inhibition of this xenograft with cisplatin and BMN 673 treatments, respectively. Cisplatin and BMN 673 treatments reduced cellular proliferation and induced apoptosis. Our findings support a personalized treatment approach for BRCA2-associated PDAC.
Assuntos
Genes BRCA2 , Neoplasias Pancreáticas/tratamento farmacológico , Ftalazinas/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Técnicas de Silenciamento de Genes , Mutação em Linhagem Germinativa , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/genéticaRESUMO
PTP-PEST is a cytosolic ubiquitous protein tyrosine phosphatase (PTP) that contains, in addition to its catalytic domain, several protein-protein interaction domains that allow it to interface with several signaling pathways. Among others, PTP-PEST is a key regulator of cellular motility and cytoskeleton dynamics. The complexity of the PTP-PEST interactome underscores the necessity to identify its interacting partners and physiological substrates in order to further understand its role in focal adhesion complex turnover and actin organization. Using a modified yeast substrate trapping two-hybrid system, we identified a cytosolic adaptor protein named Src kinase-associated phosphoprotein 55 homologue (SKAP-Hom) as a novel substrate of PTP-PEST. To confirm PTP-PEST interaction with SKAP-Hom, in vitro pull down assays were performed demonstrating that the PTP catalytic domain and Proline-rich 1 (P1) domain are respectively binding to the SKAP-Hom Y260 and Y297 residues and its SH3 domain. Subsequently, we generated and rescued SKAP-Hom-deficient mouse embryonic fibroblasts (MEFs) with WT SKAP-Hom, SKAP-Hom tyrosine mutants (Y260F, Y260F/Y297F), or SKAP-Hom SH3 domain mutant (W335K). Given the role of PTP-PEST, wound-healing and trans-well migration assays were performed using the generated lines. Indeed, SKAP-Hom-deficient MEFs showed a defect in migration compared with WT-rescued MEFs. Interestingly, the SH3 domain mutant-rescued MEFs showed an enhanced cell migration corresponding potentially with higher tyrosine phosphorylation levels of SKAP-Hom. These findings suggest a novel role of SKAP-Hom and its phosphorylation in the regulation of cellular motility. Moreover, these results open new avenues by which PTP-PEST regulates cellular migration, a hallmark of metastasis.
Assuntos
Movimento Celular/fisiologia , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 12/metabolismo , Substituição de Aminoácidos , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos Knockout , Mutação de Sentido Incorreto , Proteína Tirosina Fosfatase não Receptora Tipo 12/genética , Técnicas do Sistema de Duplo-Híbrido , Domínios de Homologia de srcRESUMO
Lrp5/6 are crucial coreceptors for Wnt/beta-catenin signaling, a pathway biochemically distinct from noncanonical Wnt signaling pathways. Here, we examined the possible participation of Lrp5/6 in noncanonical Wnt signaling. We found that Lrp6 physically interacts with Wnt5a, but that this does not lead to phosphorylation of Lrp6 or activation of the Wnt/beta-catenin pathway. Overexpression of Lrp6 blocks activation of the Wnt5a downstream target Rac1, and this effect is dependent on intact Lrp6 extracellular domains. These results suggested that the extracellular domain of Lrp6 inhibits noncanonical Wnt signaling in vitro. In vivo, Lrp6-/- mice exhibited exencephaly and a heart phenotype. Surprisingly, these defects were rescued by deletion of Wnt5a, indicating that the phenotypes resulted from noncanonical Wnt gain-of-function. Similarly, Lrp5 and Lrp6 antisense morpholino-treated Xenopus embryos exhibited convergent extension and heart phenotypes that were rescued by knockdown of noncanonical XWnt5a and XWnt11. Thus, we provide evidence that the extracellular domains of Lrp5/6 behave as physiologically relevant inhibitors of noncanonical Wnt signaling during Xenopus and mouse development in vivo.
Assuntos
Proteínas Relacionadas a Receptor de LDL/química , Proteínas Relacionadas a Receptor de LDL/metabolismo , Receptores de LDL/química , Receptores de LDL/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Deleção de Genes , Coração/embriologia , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/metabolismo , Heterozigoto , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Camundongos Mutantes , Defeitos do Tubo Neural/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Fenótipo , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Receptores de LDL/deficiência , Transdução de Sinais/efeitos dos fármacos , Proteína Wnt-5a , Xenopus/embriologia , Xenopus/metabolismo , beta Catenina/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The floor plate (FP), a signaling center and a structure rich in radial glia-like cells, has been traditionally thought to be devoid of neurons and neuronal progenitors. However, in the midbrain, the FP contains neurons of the dopaminergic (DA) lineage that require contact with radial glia-like cells for their induction. We, therefore, decided to explore the interaction relationship between radial glia and neurons during DA neurogenesis. Taking advantage of a novel FP radial glia-like cell culture system and retroviruses, DA neurons were lineage traced in vitro. In utero BrdU pulse-chases extensively labeled the midbrain FP and traced DA neurons both in vivo and in FP cultures. Moreover, from E9.5 to E13.5 the midbrain FP contained dividing cells only in the most apical part of the neuroepithelium, in cells identified as radial glia-like cells. We, therefore, hypothesized that midbrain FP radial glia-like cells could be DA progenitors and tested our hypothesis in vivo. Lineage tracing of DA progenitors with EGFP in Tis21-EGFP knock-in mice, and genetic fate mapping in GLAST::CreERT2/ZEG mice identified the neuroepithelium of the midbrain FP, and specifically, GLAST+ radial glia-like cells as DA progenitors. Combined, our experiments support the concept that the midbrain FP differs from other FP regions and demonstrate that FP radial glia-like cells in the midbrain are neurogenic and give rise to midbrain DA neurons.
Assuntos
Padronização Corporal/fisiologia , Dopamina/metabolismo , Células-Tronco Embrionárias/citologia , Mesencéfalo/citologia , Neuroglia/metabolismo , Fatores Etários , Animais , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/genética , Bromodesoxiuridina/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Embrião de Mamíferos , Antagonistas de Estrogênios/farmacologia , Transportador 1 de Aminoácido Excitatório/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Genes Supressores de Tumor , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Proteínas Imediatamente Precoces/genética , Mesencéfalo/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuroglia/efeitos dos fármacos , Gravidez , Tamoxifeno/farmacologia , Proteínas Supressoras de TumorRESUMO
Proper dialogue between presynaptic neurons and their targets is essential for correct synaptic assembly and function. At central synapses, Wnt proteins function as retrograde signals to regulate axon remodeling and the accumulation of presynaptic proteins. Loss of Wnt7a function leads to defects in the localization of presynaptic markers and in the morphology of the presynaptic axons. We show that loss of function of Dishevelled-1 (Dvl1) mimics and enhances the Wnt7a phenotype in the cerebellum. Although active zones appear normal, electrophysiological recordings in cerebellar slices from Wnt7a/Dvl1 double mutant mice reveal a defect in neurotransmitter release at mossy fiber-granule cell synapses. Deficiency in Dvl1 decreases, whereas exposure to Wnt increases, synaptic vesicle recycling in mossy fibers. Dvl increases the number of Bassoon clusters, and like other components of the Wnt pathway, it localizes to synaptic sites. These findings demonstrate that Wnts signal across the synapse on Dvl-expressing presynaptic terminals to regulate synaptic assembly and suggest a potential novel function for Wnts in neurotransmitter release.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Neurotransmissores/metabolismo , Fosfoproteínas/fisiologia , Terminações Pré-Sinápticas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Transdução de Sinais/fisiologia , Sinapses/fisiologia , Proteínas Wnt/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células Cultivadas , Proteínas Desgrenhadas , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fenótipo , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Sinapses/metabolismo , Sinapses/ultraestrutura , Proteínas Wnt/deficiência , Proteínas Wnt/genéticaRESUMO
Mutations in known breast cancer susceptibility genes account for a minority of the familial aggregation of the disease. To search for further breast cancer susceptibility genes, we performed a combined analysis of four genome-wide linkage screens, which included a total of 149 multiple case breast cancer families. All families included at least three cases of breast cancer diagnosed below age 60 years, at least one of whom had been tested and found not to carry a BRCA1 or BRCA2 mutation. Evidence for linkage was assessed using parametric linkage analysis, assuming both a dominant and a recessive mode of inheritance, and using nonparametric methods. The highest LOD score obtained in any analysis of the combined data was 1.80 under the dominant model, in a region on chromosome 4 close to marker D4S392. Three further LOD scores over 1 were identified in the parametric analyses and two in the nonparametric analyses. A maximum LOD score of 2.40 was found on chromosome arm 2p in families with four or more cases of breast cancer diagnosed below age 50 years. The number of linkage peaks did not differ from the number expected by chance. These results suggest regions that may harbor novel breast cancer susceptibility genes. They also indicate that no single gene is likely to account for a large fraction of the familial aggregation of breast cancer that is not due to mutations in BRCA1 or BRCA2.
Assuntos
Neoplasias da Mama/genética , Ligação Genética , Predisposição Genética para Doença , Genoma Humano , Feminino , Genes BRCA1 , Genes BRCA2 , Testes Genéticos , Humanos , Escore Lod , Masculino , Modelos EstatísticosRESUMO
Midbrain neurons synthesizing the neurotransmitter dopamine play a central role in the modulation of different brain functions and are associated with major neurological and psychiatric disorders. Despite the importance of these cells, the molecular mechanisms controlling their development are still poorly understood. The secreted glycoprotein Wnt1 is expressed in close vicinity to developing midbrain dopaminergic neurons. Here, we show that Wnt1 regulates the genetic network, including Otx2 and Nkx2-2, that is required for the establishment of the midbrain dopaminergic progenitor domain during embryonic development. In addition, Wnt1 is required for the terminal differentiation of midbrain dopaminergic neurons at later stages of embryogenesis. These results identify Wnt1 as a key molecule in the development of midbrain dopaminergic neurons in vivo. They also suggest the Wnt1-controlled signaling pathway as a promising target for new therapeutic strategies in the treatment of Parkinson's disease.
Assuntos
Diferenciação Celular/fisiologia , Mesencéfalo/embriologia , Neurônios/fisiologia , Transdução de Sinais/fisiologia , Células-Tronco/fisiologia , Proteína Wnt1/metabolismo , Animais , Proteína Homeobox Nkx-2.2 , Imuno-Histoquímica , Hibridização In Situ , Mesencéfalo/metabolismo , Camundongos , Camundongos Transgênicos , Microesferas , Neurônios/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismoRESUMO
Cultures of three-dimensional aggregates of embryonic stem cells (ESCs) called embryoid bodies (EBs) provide a valuable system for analyzing molecular mechanisms that regulate differentiation of this unique cell type. Cyclin-dependent kinase inhibitor p27Kip1 (p27) becomes elevated during the differentiation of mouse ESCs (mESCs). In this study, various aspects of differentiation of EBs produced from normal and p27-deficient mESCs were analyzed to address the biological significance of this elevation. It was found that EBs lacking p27 grew significantly bigger, but this was not accompanied by detect-able abnormalities in the activities of cyclin-dependent kinases (CDKs). In most EB cells, downregulation of activating cyclins rather than upregulation of inhibiting p27 is probably responsible for lowering the activity of their CDKs. Abnormalities in the development of specific cell lineages were also observed in p27-deficient EBs. These included elimination of cells positive for cytokeratin endo-A (TROMA-I) and increased proliferation and formation of cavities originating from cells positive for Lewis-X. Our data also suggest that although two different pools of Lewis-X-expressing cells, cluster forming (ESC-like) and cavity forming (neural progenitors), normally exist in EBs, the absence of p27 leads to the enhancement of only the neural pool. No failure was found when the neurogenic capacity of p27-deficient mESCs was tested using various protein markers. Together, our data point to a dual role of p27 in mESCs, with one role being in the regulation of proliferation and the other role in establishing some other aspects of a differentiated phenotype.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Embrião de Mamíferos/fisiologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologia , Animais , Western Blotting , Ciclo Celular , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p27 , Regulação para Baixo , Citometria de Fluxo , Imuno-Histoquímica , Imunoprecipitação , Antígenos CD15/metabolismo , Camundongos , Microscopia de Fluorescência , Neurônios/metabolismo , Fenótipo , Células-Tronco/metabolismo , Fatores de Tempo , Regulação para CimaRESUMO
The transcription factor TFIID consists of TATA-binding protein (TBP) and TBP-associated factors (TAFs). TAFs are essential for modulation of transcriptional activity but the regulation of TAFs is complex and many important aspects remain unclear. In this study, we have identified and characterized five novel truncated forms of the TFIID subunit TAF4 (TAF(II)135). Analysis of the mouse gene structure revealed that all truncations were the results of alternative splicing and resulted in the loss of domains or parts of domains implicated in TAF4 functional interactions. Results from transcriptional assays showed that several of the TAF4 isoforms exerted dominant negative effects on TAF4 activity in nuclear receptor-mediated transcriptional activation. In addition, alternative TAF4 isoforms could be detected in specific cell types. Our results indicate an additional level of complexity in TAF4-mediated regulation of transcription and suggest context-specific roles for these new TAF4 isoforms in transcriptional regulation in vivo.
Assuntos
Receptores do Ácido Retinoico/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/química , Fator de Transcrição TFIID/fisiologia , Processamento Alternativo , Animais , Encéfalo/metabolismo , Células COS , Células Cultivadas , Chlorocebus aethiops , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Deleção de Genes , Expressão Gênica , Componentes Genômicos/genética , Camundongos , Camundongos Endogâmicos BALB C , Neurônios/metabolismo , Isoformas de Proteínas , Subunidades Proteicas , Receptores do Ácido Retinoico/fisiologia , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genética , Transcrição GênicaRESUMO
Valproate (VPA) and lithium have been used for many years in the treatment of manic depression. However, their mechanisms of action remain poorly understood. Recent studies suggest that lithium and VPA inhibit GSK-3beta, a serine/threonine kinase involved in the insulin and WNT signaling pathways. Inhibition of GSK-3beta by high concentrations of lithium has been shown to mimic WNT-7a signaling by inducing axonal remodeling and clustering of synapsin I in developing neurons. Here we have compared the effect of therapeutic concentrations of lithium and VPA during neuronal maturation. VPA and, to a lesser extent, lithium induce clustering of synapsin I. In addition, lithium and VPA induce similar changes in the morphology of axons by increasing growth cone size, spreading, and branching. More importantly, both mood stabilizers decrease the level of MAP-1B-P, a GSK-3beta-phosphorylated form of MAP-1B in developing neurons, suggesting that therapeutic concentrations of these mood stabilizers inhibit GSK-3beta. In vitro kinase assays show that therapeutic concentrations of VPA do not inhibit GSK-3beta but that therapeutic concentrations of lithium partially inhibit GSK-3beta activity. Our results support the idea that both mood stabilizers inhibit GSK-3beta in developing neurons through different pathways. Lithium directly inhibits GSK-3beta in contrast to VPA, which inhibits GSK-3beta indirectly by an as-yet-unknown pathway. These findings may have important implications for the development of new strategies to treat bipolar disorders.