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1.
J Immunother Cancer ; 9(7)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34285105

RESUMO

M2 macrophages promote tumor progression and therapy resistance, whereas proimmunogenic M1 macrophages can contribute to the efficacy of cytostatic and immunotherapeutic strategies. The abundance of M2 macrophages in the immune infiltrate of many cancer types has prompted the search for strategies to target and eliminate this subset. From our prior experiments in syngeneic mouse tumor models, we learned that pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) did not merely result in tumor cell death, but also in the modulation of the tumor immune infiltrate. This included a prominent decrease in the numbers of macrophages as well as an increase in the M1/M2 macrophage ratio. Investigation of the mechanism underlying this finding in primary murine macrophage cultures revealed that M2 macrophages are significantly more sensitive to MEK inhibition-induced cell death than their M1 counterparts. Further analyses showed that the p38 MAPK pathway, which is activated in M1 macrophages only, renders these cells resistant to death by MEK inhibition. In conclusion, the dependency of M2 macrophages on the MEK/extracellular-signal regulated kinase (ERK) pathway empowers MEK inhibitors to selectively eliminate this subset from the tumor microenvironment.

2.
Pain ; 162(7): 2070-2086, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33492035

RESUMO

ABSTRACT: After surgery, acute pain is still managed insufficiently and may lead to short-term and long-term complications including chronic postsurgical pain and an increased prescription of opioids. Thus, identifying new targets specifically implicated in postoperative pain is of utmost importance to develop effective and nonaddictive analgesics. Here, we used an integrated and multimethod workflow to reveal unprecedented insights into proteome dynamics in dorsal root ganglia (DRG) of mice after plantar incision (INC). Based on a detailed characterization of INC-associated pain-related behavior profiles, including a novel paradigm for nonevoked pain, we performed quantitative mass-spectrometry-based proteomics in DRG 1 day after INC. Our data revealed a hitherto unknown INC-regulated protein signature in DRG with changes in distinct proteins and cellular signaling pathways. In particular, we show the differential regulation of 44 protein candidates, many of which are annotated with pathways related to immune and inflammatory responses such as MAPK/extracellular signal-regulated kinases signaling. Subsequent orthogonal assays comprised multiplex Western blotting, bioinformatic protein network analysis, and immunolabeling in independent mouse cohorts to validate (1) the INC-induced regulation of immune/inflammatory pathways and (2) the high priority candidate Annexin A1. Taken together, our results propose novel potential targets in the context of incision and, therefore, represent a highly valuable resource for further mechanistic and translational studies of postoperative pain.


Assuntos
Dor Aguda , Gânglios Espinais , Animais , Camundongos , Dor Pós-Operatória , Proteoma , Ratos , Ratos Sprague-Dawley
3.
Lab Invest ; 100(10): 1288-1299, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32601356

RESUMO

Histomorphology and immunohistochemistry are the most common ways of cancer classification in routine cancer diagnostics, but often reach their limits in determining the organ origin in metastasis. These cancers of unknown primary, which are mostly adenocarcinomas or squamous cell carcinomas, therefore require more sophisticated methodologies of classification. Here, we report a multiplex protein profiling-based approach for the classification of fresh frozen and formalin-fixed paraffin-embedded (FFPE) cancer tissue samples using the digital western blot technique DigiWest. A DigiWest-compatible FFPE extraction protocol was developed, and a total of 634 antibodies were tested in an initial set of 16 FFPE samples covering tumors from different origins. Of the 303 detected antibodies, 102 yielded significant correlation of signals in 25 pairs of fresh frozen and FFPE primary tumor samples, including head and neck squamous cell carcinomas (HNSC), lung squamous cell carcinomas (LUSC), lung adenocarcinomas (LUAD), colorectal adenocarcinomas (COAD), and pancreatic adenocarcinomas (PAAD). For this signature of 102 analytes (covering 88 total proteins and 14 phosphoproteins), a support vector machine (SVM) algorithm was developed. This allowed for the classification of the tissue of origin for all five tumor types studied here with high overall accuracies in both fresh frozen (90.4%) and FFPE (77.6%) samples. In addition, the SVM classifier reached an overall accuracy of 88% in an independent validation cohort of 25 FFPE tumor samples. Our results indicate that DigiWest-based protein profiling represents a valuable method for cancer classification, yielding conclusive and decisive data not only from fresh frozen specimens but also FFPE samples, thus making this approach attractive for routine clinical applications.


Assuntos
Western Blotting/métodos , Neoplasias/classificação , Análise Serial de Proteínas/métodos , Algoritmos , Biomarcadores Tumorais/metabolismo , Western Blotting/estatística & dados numéricos , Criopreservação , Formaldeído , Humanos , Proteínas de Neoplasias/metabolismo , Neoplasias/diagnóstico , Neoplasias/metabolismo , Especificidade de Órgãos , Inclusão em Parafina , Análise Serial de Proteínas/estatística & dados numéricos , Máquina de Vetores de Suporte , Fixação de Tecidos
4.
Biochim Biophys Acta Mol Cell Res ; 1867(8): 118722, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32302667

RESUMO

Dermal fibroblasts seem critical for epidermal maturation and differentiation and recent work demonstrated that diseased fibroblasts may drive pathophysiological processes. Nevertheless, still very little is known about the actual crosstalk between epidermal keratinocytes and dermal fibroblasts and the impact of dermal fibroblasts on epidermal maturation and differentiation. Aiming for a more fundamental understanding of the impact of the cellular crosstalk between keratinocytes and fibroblasts on the skin homeostasis, we generated full-thickness skin equivalents with and without fibroblasts and subsequently analysed them for the expression of skin differentiation markers, their barrier function, skin lipid content and epidermal cell signalling. Skin equivalents without fibroblasts consistently showed an impaired differentiation and dysregulated expression of skin barrier and tight junction proteins, increased skin permeability, and a decreased skin lipid/protein ratio. Most interestingly, impaired Ras/Raf/ERK/MEK signalling was evident in skin equivalents without fibroblasts. Our data clearly indicate that the epidermal-dermal crosstalk between keratinocytes and fibroblasts is critical for adequate skin differentiation and that fibroblasts orchestrate epidermal differentiation processes.


Assuntos
Células Epidérmicas/metabolismo , Fibroblastos/metabolismo , Homeostase/fisiologia , Queratinócitos/metabolismo , Pele/metabolismo , Diferenciação Celular , Células Epidérmicas/patologia , Epiderme/metabolismo , Homeostase/genética , Humanos , Queratinócitos/patologia , Permeabilidade , Pele/patologia , Absorção Cutânea
5.
Nat Commun ; 10(1): 2197, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097693

RESUMO

In colorectal cancer (CRC), aberrant Wnt signalling is essential for tumorigenesis and maintenance of cancer stem cells. However, how other oncogenic pathways converge on Wnt signalling to modulate stem cell homeostasis in CRC currently remains poorly understood. Using large-scale compound screens in CRC, we identify MEK1/2 inhibitors as potent activators of Wnt/ß-catenin signalling. Targeting MEK increases Wnt activity in different CRC cell lines and murine intestine in vivo. Truncating mutations of APC generated by CRISPR/Cas9 strongly synergize with MEK inhibitors in enhancing Wnt responses in isogenic CRC models. Mechanistically, we demonstrate that MEK inhibition induces a rapid downregulation of AXIN1. Using patient-derived CRC organoids, we show that MEK inhibition leads to increased Wnt activity, elevated LGR5 levels and enrichment of gene signatures associated with stemness and cancer relapse. Our study demonstrates that clinically used MEK inhibitors inadvertently induce stem cell plasticity, revealing an unknown side effect of RAS pathway inhibition.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Células-Tronco Neoplásicas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Antineoplásicos/uso terapêutico , Biópsia , Sistemas CRISPR-Cas/genética , Carcinogênese/efeitos dos fármacos , Linhagem Celular Tumoral , Plasticidade Celular/efeitos dos fármacos , Neoplasias Colorretais/patologia , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Intestinos/citologia , Intestinos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteômica , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/metabolismo
6.
Sci Rep ; 9(1): 7258, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-31076619

RESUMO

Pooled human platelet lysate (pHPL) is increasingly used as replacement of animal serum for manufacturing of stromal cell therapeutics. Porcine heparin is commonly applied to avoid clotting of pHPL-supplemented medium but the influence of heparin on cell behavior is still unclear. Aim of this study was to investigate cellular uptake of heparin by fluoresceinamine-labeling and its impact on expression of genes, proteins and function of human stromal cells derived from bone marrow (BM), umbilical cord (UC) and white adipose tissue (WAT). Cells were isolated and propagated using various pHPL-supplemented media with or without heparin. Flow cytometry and immunocytochemistry showed differential cellular internalization and lysosomal accumulation of heparin. Transcriptome profiling revealed regulation of distinct gene sets by heparin including signaling cascades involved in proliferation, cell adhesion, apoptosis, inflammation and angiogenesis, depending on stromal cell origin. The influence of heparin on the WNT, PDGF, NOTCH and TGFbeta signaling pathways was further analyzed by a bead-based western blot revealing most alterations in BM-derived stromal cells. Despite these observations heparin had no substantial effect on long-term proliferation and in vitro tri-lineage differentiation of stromal cells, indicating compatibility for clinically applied cell products.


Assuntos
Expressão Gênica/fisiologia , Heparina/metabolismo , Células Estromais/metabolismo , Plaquetas/metabolismo , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Humanos , Soro/metabolismo , Transdução de Sinais/fisiologia , Cordão Umbilical/metabolismo
7.
PLoS Genet ; 15(3): e1008076, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30925167

RESUMO

Organoid cultures derived from colorectal cancer (CRC) samples are increasingly used as preclinical models for studying tumor biology and the effects of targeted therapies under conditions capturing in vitro the genetic make-up of heterogeneous and even individual neoplasms. While 3D cultures are initiated from surgical specimens comprising multiple cell populations, the impact of tumor heterogeneity on drug effects in organoid cultures has not been addressed systematically. Here we have used a cohort of well-characterized CRC organoids to study the influence of tumor heterogeneity on the activity of the KRAS/MAPK-signaling pathway and the consequences of treatment by inhibitors targeting EGFR and downstream effectors. MAPK signaling, analyzed by targeted proteomics, shows unexpected heterogeneity irrespective of RAS mutations and is associated with variable responses to EGFR inhibition. In addition, we obtained evidence for intratumoral heterogeneity in drug response among parallel "sibling" 3D cultures established from a single KRAS-mutant CRC. Our results imply that separate testing of drug effects in multiple subpopulations may help to elucidate molecular correlates of tumor heterogeneity and to improve therapy response prediction in patients.


Assuntos
Técnicas de Cultura de Células/métodos , Neoplasias Colorretais/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Linhagem Celular Tumoral , Estudos de Coortes , Neoplasias Colorretais/fisiopatologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Genes erbB-1 , Humanos , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Mutação , Organoides/metabolismo , Organoides/fisiologia , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Transdução de Sinais , Proteínas ras/genética
8.
J Extracell Vesicles ; 6(1): 1378056, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29184623

RESUMO

Extracellular vesicles (EVs) are membrane particles secreted from cells into all body fluids. Several EV populations exist differing in size and cellular origin. Using differential centrifugation EVs pelleting at 14,000 g ("microvesicles" (MV)) and 100,000 g ("exosomes") are distinguishable by protein markers. Neutral sphingomyelinase (nSMase) inhibition has been shown to inhibit exosome release from cells and has since been used to study their functional implications. How nSMases (also known as SMPD2 and SMPD3) affect the basal secretion of MVs is unclear. Here we investigated how SMPD2/3 impact both EV populations. SMPD2/3 inhibition by GW4869 or RNAi decreases secretion of exosomes, but also increases secretion of MVs from the plasma membrane. Both populations differ significantly in metabolite composition and Wnt proteins are specifically loaded onto MVs under these conditions. Taken together, our data reveal a novel regulatory function of SMPD2/3 in vesicle budding from the plasma membrane and clearly suggest that - despite the different vesicle biogenesis - the routes of vesicular export are adaptable.

9.
Sci Signal ; 10(461)2017 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-28074006

RESUMO

Wnt signaling plays an important role in the self-renewal and differentiation of stem cells. The secretion of Wnt ligands requires Evi (also known as Wls). Genetically ablating Evi provides an experimental approach to studying the consequence of depleting all redundant Wnt proteins, and overexpressing Evi enables a nonspecific means of increasing Wnt signaling. We generated Evi-deficient and Evi-overexpressing mouse embryonic stem cells (ESCs) to analyze the role of autocrine Wnt production in self-renewal and differentiation. Self-renewal was reduced in Evi-deficient ESCs and increased in Evi-overexpressing ESCs in the absence of leukemia inhibitory factor, which supports the self-renewal of ESCs. The differentiation of ESCs into cardiomyocytes was enhanced when Evi was overexpressed and teratoma formation and growth of Evi-deficient ESCs in vivo were impaired, indicating that autocrine Wnt ligands were necessary for ESC differentiation and survival. ESCs lacking autocrine Wnt signaling had mitotic defects and showed genomic instability. Together, our study demonstrates that autocrine Wnt secretion is important for the survival, chromosomal stability, differentiation, and tumorigenic potential of ESCs.


Assuntos
Comunicação Autócrina , Proliferação de Células/genética , Instabilidade Genômica , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Wnt/genética , Animais , Diferenciação Celular/genética , Autorrenovação Celular , Sobrevivência Celular/genética , Células Cultivadas , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Embrionárias Murinas/transplante , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt
10.
PLoS One ; 11(6): e0155999, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27248690

RESUMO

Cellular signalling pathways consolidate multiple molecular interactions into working models of signal propagation, amplification, and modulation. They are described and visualized as networks. Adjusting network topologies to experimental data is a key goal of systems biology. While network reconstruction algorithms like nested effects models are well established tools of computational biology, their data requirements can be prohibitive for their practical use. In this paper we suggest focussing on well defined aspects of a pathway and develop the computational tools to do so. We adapt the framework of nested effect models to focus on a specific aspect of activated Wnt signalling in HCT116 colon cancer cells: Does the activation of Wnt target genes depend on the secretion of Wnt ligands or do mutations in the signalling molecule ß-catenin make this activation independent from them? We framed this question into two competing classes of models: Models that depend on Wnt ligands secretion versus those that do not. The model classes translate into restrictions of the pathways in the network topology. Wnt dependent models are more flexible than Wnt independent models. Bayes factors are the standard Bayesian tool to compare different models fairly on the data evidence. In our analysis, the Bayes factors depend on the number of potential Wnt signalling target genes included in the models. Stability analysis with respect to this number showed that the data strongly favours Wnt ligands dependent models for all realistic numbers of target genes.


Assuntos
Modelos Teóricos , Algoritmos , Teorema de Bayes , Linhagem Celular Tumoral , Humanos , Transdução de Sinais , Proteínas Wnt/metabolismo
11.
Cancer Metab ; 3: 11, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26500770

RESUMO

BACKGROUND: Numerous studies have demonstrated that functional mitochondria are required for tumorigenesis, suggesting that mitochondrial oxidative phosphorylation (OXPHOS) might be a potential target for cancer therapy. In this study, we investigated the effects of BAY 87-2243, a small molecule that inhibits the first OXPHOS enzyme (complex I), in melanoma in vitro and in vivo. RESULTS: BAY 87-2243 decreased mitochondrial oxygen consumption and induced partial depolarization of the mitochondrial membrane potential. This was associated with increased reactive oxygen species (ROS) levels, lowering of total cellular ATP levels, activation of AMP-activated protein kinase (AMPK), and reduced cell viability. The latter was rescued by the antioxidant vitamin E and high extracellular glucose levels (25 mM), indicating the involvement of ROS-induced cell death and a dependence on glycolysis for cell survival upon BAY 87-2243 treatment. BAY 87-2243 significantly reduced tumor growth in various BRAF mutant melanoma mouse xenografts and patient-derived melanoma mouse models. Furthermore, we provide evidence that inhibition of mutated BRAF using the specific small molecule inhibitor vemurafenib increased the OXPHOS dependency of BRAF mutant melanoma cells. As a consequence, the combination of both inhibitors augmented the anti-tumor effect of BAY 87-2243 in a BRAF mutant melanoma mouse xenograft model. CONCLUSIONS: Taken together, our results suggest that complex I inhibition has potential clinical applications as a single agent in melanoma and also might be efficacious in combination with BRAF inhibitors in the treatment of patients with BRAF mutant melanoma.

12.
Genome Med ; 7(1): 46, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26120366

RESUMO

Mesenchymal stem cells (MSCs) are promising candidates for cellular therapies ranging from tissue repair in regenerative medicine to immunomodulation in graft versus host disease after allogeneic transplantation or in autoimmune diseases. Nonetheless, progress has been hampered by their enormous phenotypic as well as functional heterogeneity and the lack of uniform standards and guidelines for quality control. In this study, we describe a method to perform cellular phenotyping by high-throughput RNA interference in primary human bone marrow MSCs. We have shown that despite heterogeneity of MSC populations, robust functional assays can be established that are suitable for high-throughput and high-content screening. We profiled primary human MSCs against human fibroblasts. Network analysis showed a kinome fingerprint that differs from human primary fibroblasts as well as fibroblast cell lines. In conclusion, this study shows that high-throughput screening in primary human MSCs can be reliably used for kinome fingerprinting.

13.
PLoS One ; 10(5): e0127146, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26010451

RESUMO

Next generation sequencing (NGS) is an emerging technology becoming relevant for genotyping of clinical samples. Here, we assessed the stability of amplicon sequencing from formalin-fixed paraffin-embedded (FFPE) and paired frozen samples from colorectal cancer metastases with different analysis pipelines. 212 amplicon regions in 48 cancer related genes were sequenced with Illumina MiSeq using DNA isolated from resection specimens from 17 patients with colorectal cancer liver metastases. From ten of these patients, paired fresh frozen and routinely processed FFPE tissue was available for comparative study. Sample quality of FFPE tissues was determined by the amount of amplifiable DNA using qPCR, sequencing libraries were evaluated using Bioanalyzer. Three bioinformatic pipelines were compared for analysis of amplicon sequencing data. Selected hot spot mutations were reviewed using Sanger sequencing. In the sequenced samples from 16 patients, 29 non-synonymous coding mutations were identified in eleven genes. Most frequent were mutations in TP53 (10), APC (7), PIK3CA (3) and KRAS (2). A high concordance of FFPE and paired frozen tissue samples was observed in ten matched samples, revealing 21 identical mutation calls and only two mutations differing. Comparison of these results with two other commonly used variant calling tools, however, showed high discrepancies. Hence, amplicon sequencing can potentially be used to identify hot spot mutations in colorectal cancer metastases in frozen and FFPE tissue. However, remarkable differences exist among results of different variant calling tools, which are not only related to DNA sample quality. Our study highlights the need for standardization and benchmarking of variant calling pipelines, which will be required for translational and clinical applications.


Assuntos
Neoplasias Colorretais/genética , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Proteínas de Neoplasias/genética , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias Colorretais/metabolismo , Feminino , Humanos , Masculino , Proteínas de Neoplasias/metabolismo
14.
Nat Commun ; 4: 2610, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24162018

RESUMO

Aberrant regulation of the Wnt/ß-catenin pathway has an important role during the onset and progression of colorectal cancer, with over 90% of cases of sporadic colon cancer featuring mutations in APC or ß-catenin. However, it has remained a point of controversy whether these mutations are sufficient to activate the pathway or require additional upstream signals. Here we show that colorectal tumours express elevated levels of Wnt3 and Evi/Wls/GPR177. We found that in colon cancer cells, even in the presence of mutations in APC or ß-catenin, downstream signalling remains responsive to Wnt ligands and receptor proximal signalling. Furthermore, we demonstrate that truncated APC proteins bind ß-catenin and key components of the destruction complex. These results indicate that cells with mutations in APC or ß-catenin depend on Wnt ligands and their secretion for a sufficient level of ß-catenin signalling, which potentially opens new avenues for therapeutic interventions by targeting Wnt secretion via Evi/Wls.


Assuntos
Adenocarcinoma/genética , Proteína da Polipose Adenomatosa do Colo/genética , Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Receptores Acoplados a Proteínas G/genética , Proteína Wnt3/genética , beta Catenina/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Mutação , Transplante de Neoplasias , Receptor EphB2/genética , Receptor EphB2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Proteína Wnt3/metabolismo , beta Catenina/metabolismo
15.
Cell Rep ; 4(6): 1224-34, 2013 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-24035388

RESUMO

Wnt/ß-catenin signaling plays an important role in embryonic development and adult tissue homeostasis. When Wnt ligands bind to the receptor complex, LRP5/6 coreceptors are activated by phosphorylation and concomitantly endocytosed. In vertebrates, Wnt ligands induce caveolin-dependent endocytosis of LRP6 to relay signal downstream, whereas antagonists such as Dickkopf promote clathrin-dependent endocytosis, leading to inhibition. However, little is known about how LRP6 is directed to different internalization mechanisms, and how caveolin-dependent endocytosis is mediated. In an RNAi screen, we identified the Rab GTPase RAB8B as being required for Wnt/ß-catenin signaling. RAB8B depletion reduces LRP6 activity, ß-catenin accumulation, and induction of Wnt target genes, whereas RAB8B overexpression promotes LRP6 activity and internalization and rescues inhibition of caveolar endocytosis. In Xenopus laevis and Danio rerio, RAB8B morphants show lower Wnt activity during embryonic development. Our results implicate RAB8B as an essential evolutionary conserved component of Wnt/ß-catenin signaling through regulation of LRP6 activity and endocytosis.


Assuntos
Endocitose/fisiologia , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Wnt/genética , Animais , Células HEK293 , Células HeLa , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteínas Oncogênicas/genética , Fosforilação , Transdução de Sinais , Transfecção , Proteínas Wnt/metabolismo , Xenopus , Peixe-Zebra , Proteínas rab de Ligação ao GTP
16.
Biotechnol J ; 7(6): 768-78, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22653826

RESUMO

High-throughput RNAi or small molecule screens have proven to be powerful methodologies for the systematic dissection of cellular processes. In model organisms and cell lines, large-scale screens have identified key components of many cellular pathways and helped to identify novel targets in disease-relevant pathways. Image-based high-content screening has become an increasingly important tool in high-throughput screening, enabling changes in phenotype characteristics, such as cell morphology and cell differentiation, to be monitored. In this review, we discuss the use of image-based screening approaches to explore the behavior of adult, embryonic, and induced pluripotent stem cells. First, we review how current pluripotency and differentiation assays can be adapted to high-throughput formats. We then describe general aspects of image-based screening of cells and present an outlook on challenges for screening stem cells.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Células-Tronco/citologia , Células-Tronco/fisiologia , Animais , Diferenciação Celular/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Processamento de Imagem Assistida por Computador , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia
17.
PLoS One ; 6(12): e28338, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22162763

RESUMO

Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%-58.7±14.4% when two indicators were combined and 40-48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost.


Assuntos
Interferência de RNA , Trifosfato de Adenosina/química , Benzimidazóis/farmacologia , Sobrevivência Celular , Citoplasma/metabolismo , Reações Falso-Positivas , Fluoresceínas/farmacologia , Corantes Fluorescentes/farmacologia , Células HEK293 , Humanos , Fenótipo , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos Testes , Projetos de Pesquisa
18.
Biotechnol J ; 5(4): 368-76, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20349460

RESUMO

RNA interference (RNAi) has become a powerful tool to dissect cellular pathways and characterize gene functions. The availability of genome-wide RNAi libraries for various model organisms and mammalian cells has enabled high-throughput RNAi screenings. These RNAi screens successfully identified key components that had previously been missed in classical forward genetic screening approaches and allowed the assessment of combined loss-of-function phenotypes. Crucially, the quality of RNAi screening results depends on quantitative assays and the choice of the right biological context. In this review, we provide an overview on the design and application of high-throughput RNAi screens as well as data analysis and candidate validation strategies.


Assuntos
Marcação de Genes/métodos , Testes Genéticos/métodos , Interferência de RNA/fisiologia , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , beta Catenina/genética
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