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  • 1
    Keywords: RECEPTOR ; EXPRESSION ; INVASION ; CELL ; Germany ; KINASE ; PATHWAY ; PATHWAYS ; TYROSINE KINASE ; SYSTEM ; SYSTEMS ; GENE ; GENES ; GENOME ; PROTEIN ; PROTEINS ; validation ; BIOLOGY ; TARGET ; REQUIRES ; PCR ; SIGNALING PATHWAY ; SIGNALING PATHWAYS ; EPITHELIAL-CELLS ; systems biology ; TARGETS ; OVEREXPRESSION ; real-time PCR ; protein expression ; CROSS-TALK ; CYTOTOXICITY ; signaling ; SINGLE ; ARRAY ; analysis ; EVENTS ; technique ; USA ; quantitative ; SIGNALING NETWORK ; protein arrays ; combinatorial protein knockdown ; reverse-phase protein arrays
    Abstract: The elucidation of cross-talk events between intersecting signaling pathways is one main challenge in biological research. The complexity of protein networks, composed of different pathways, requires novel strategies and techniques to reveal relevant interrelations. Here, we established a combinatorial RNAi strategy for systematic single, double, and triple knockdown, and we measured the residual mRNAs and proteins quantitatively by quantitative real-time PCR and reverse-phase protein arrays, respectively, as a prerequisite for data analysis. Our results show that the parallel knockdown of at least three different genes is feasible while keeping both untargeted silencing and cytotoxicity low. The technique was validated by investigating the interplay of tyrosine kinase receptor ErbB2 and its downstream targets Akt-1 and MEK1 in cell invasion. This experimental approach combines multiple gene knockdown with a subsequent quantitative validation of reduced protein expression and is a major advancement toward the analysis of signaling pathways in systems biology
    Type of Publication: Journal article published
    PubMed ID: 17420474
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  • 2
    Keywords: CANCER ; EXPRESSION ; INHIBITOR ; proliferation ; SURVIVAL ; CELL ; Germany ; RISK ; GENE ; GENES ; HYBRIDIZATION ; microarray ; PROTEIN ; transcription ; TISSUE ; COMPLEX ; TRANSCRIPTION FACTOR ; IMPACT ; primary ; cell cycle ; CELL-CYCLE ; DOWN-REGULATION ; PHOSPHORYLATION ; COMPARATIVE GENOMIC HYBRIDIZATION ; PROMOTER ; UP-REGULATION ; ARRAYS ; PROMOTERS ; RATES ; MUTATIONS ; protein expression ; PROTEOMICS ; C-KIT ; RETINOBLASTOMA PROTEIN ; molecular ; ONCOLOGY ; ARRAY ; p16(INK4A) ; quantitative RT-PCR ; mRNA ; LEVEL ; TARGET GENES ; PHASE ; NUCLEAR ; LOSSES ; ENGLAND ; quantitative ; PLATFORM ; Rb ; GIST ; gastrointestinal ; DEPENDENT KINASES ; E2F1 ; E2FS ; PROGNOSTIC IMPLICATIONS ; RPPA
    Abstract: Loss of chromosome 9p is a reliable predictor of malignant behaviour in gastrointestinal stromal tumours (GISTS). p16(INK4A) located at 9p21 inhibits the CDK4/6/cyclin D complex from phosphorylating RB. Phosphorylation of RB through CDK4/6/cyclin D in early G(1) phase frees the transcription factor E2F1 from RB and enables mRNA transcription of genes essential for G(1)/S phase transition. This study aims to determine the impact of 9p loss on mRNA and protein expression of p16(INK4A) and further key cell cycle regulators in the different phases of the cell cycle. Sixty primary GISTS previously characterized for 9p loss by comparative genomic hybridization were analysed for mRNA expression of p16(INK4A), p15(INK4B), CDK4, CDK6, cyclin D, p 21(CIP1)p27(KIP1), CDK2, cyclin E, cyclin B, RB and E2F1, using quantitative RT-PCR. The protein expression of CDK6, CDK2, p2(CIP1), p27(KIP1) and phosphorylated RB (S807/S811) was evaluated using protein arrays as a novel and highly sensitive platform for profiling of protein abundance and protein phosphorylation. In parallel, the nuclear percentages of immunohistochemical staining for p16(INK4A), cyclin D, E2F1 and RB were quantified on a tissue microarray. GISTS with 9p loss had significantly higher proliferation rates, higher metastatic behaviour and shorter disease-free survival. On the molecular level, GISTS with 9p loss had a significantly reduced mRNA as well as nuclear protein expression of p16(INK4A). RB was significantly more phosphorylated in these tumours, together with increased mRNA expression and nuclear staining for E2F1 Furthermore, GISTS with 9p loss had up-regulation of the late G(1)/S phase promoters CDK2 and cyclin E. We conclude that loss of 9p accompanied by early G(1) phase inhibitor p16(INK4A) down-regulation in GISTS facilitates phosphorylation of RB, enabling F2F1-dependent transcription of genes essential for late G(1)/S phase transition. This study provides a possible basis for the accelerated proliferation and particularly malignant behaviour in GISTS with 9p loss. Copyright (C) 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd
    Type of Publication: Journal article published
    PubMed ID: 18438954
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  • 3
    Keywords: CANCER ; EXPRESSION ; tumor ; Germany ; PATHWAY ; PATHWAYS ; QUANTIFICATION ; TOOL ; GENOME ; microarray ; PROTEIN ; PROTEINS ; SAMPLE ; DRUG ; BIOLOGY ; DISCOVERY ; FIELD ; TARGET ; microarrays ; ARRAYS ; SIGNALING PATHWAY ; MASS-SPECTROMETRY ; systems biology ; CONSUMPTION ; PROTEOMICS ; protein microarray ; GASTROINTESTINAL STROMAL TUMORS ; signaling ; review ; RE ; CAPACITY ; ARRAY ; DRUG DISCOVERY ; IMMUNOSORBENT-ASSAY ELISA ; PHASE ; TECHNOLOGY ; ENGLAND ; quantitative ; PLATFORM ; TUMOR BIOLOGY ; 1PAQ ; biomarker identification ; CANCER-CELL LINES ; CODED AFFINITY TAGS ; quantitative biology ; QUANTITATIVE PROTEOMICS ; reverse phase microarray ; reverse phase protein array ; REVERSE-PHASE
    Abstract: Background: Protein microarrays have the potential to join the field of quantitative proteomics as a standard method. This antibody-based experimental platform allows the highly sensitive and highly specific quantification of selected target proteins and excels with high sample capacity. As a key feature, numerous arrays can be produced in parallel, thus minimizing sample consumption. Objective: The recent progress made in the field of reverse phase protein arrays is summarized, with a focus on the introduction of normalization measures, as introduced in the authors' infrared-based protein arrays with quantitative readout approach. Conclusion: Tumor biology as well as drug discovery applications will soon benefit from the comprehensive description of signaling pathways and protein microarrays are an appropriate tool to achieve this goal
    Type of Publication: Journal article published
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  • 4
    Keywords: Application, BIOLOGY, BREAST-CANCER, CANCER, CANCER PROGRESSION, cancer research, CELL LUNG-CANCER,
    Abstract: A detailed and quantitative analysis of disease-relevant signaling will greatly contribute to our understanding of tumorigenesis and cancer progression, and thus open new strategies for drug discovery. However, throughput and sensitivity of currently established methods available for proteome profiling do not comply with the needs of clinical research such as high sample capacity and low sample consumption. Protein microarrays emerged as a promising alternative to analyze the abundance of proteins and their phosphorylation status on a high-throughput level. Here we summarize recent methodological advancements in the field of reverse-phase protein arrays and demonstrate their potential for clinical research as well as for in vitro applications
    Type of Publication: Journal article published
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  • 5
    Abstract: Background: In breast cancer, overexpression of the transmembrane tyrosine kinase ERBB2 is an adverse prognostic marker, and occurs in almost 30% of the patients. For therapeutic intervention, ERBB2 is targeted by monoclonal antibody trastuzumab in adjuvant settings; however, de novo resistance to this antibody is still a serious issue, requiring the identification of additional targets to overcome resistance. In this study, we have combined computational simulations, experimental testing of simulation results, and finally reverse engineering of a protein interaction network to define potential therapeutic strategies for de novo trastuzumab resistant breast cancer. Results: First, we employed Boolean logic to model regulatory interactions and simulated single and multiple protein loss-of-functions. Then, our simulation results were tested experimentally by producing single and double knockdowns of the network components and measuring their effects on G1/S transition during cell cycle progression. Combinatorial targeting of ERBB2 and EGFR did not affect the response to trastuzumab in de novo resistant cells, which might be due to decoupling of receptor activation and cell cycle progression. Furthermore, examination of c-MYC in resistant as well as in sensitive cell lines, using a specific chemical inhibitor of c-MYC (alone or in combination with trastuzumab), demonstrated that both trastuzumab sensitive and resistant cells responded to c-MYC perturbation. Conclusion: In this study, we connected ERBB signaling with G1/S transition of the cell cycle via two major cell signaling pathways and two key transcription factors, to model an interaction network that allows for the identification of novel targets in the treatment of trastuzumab resistant breast cancer. Applying this new strategy, we found that, in contrast to trastuzumab sensitive breast cancer cells, combinatorial targeting of ERBB receptors or of key signaling intermediates does not have potential for treatment of de novo trastuzumab resistant cells. Instead, c-MYC was identified as a novel potential target protein in breast cancer cells
    Type of Publication: Journal article published
    PubMed ID: 19118495
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  • 6
    Keywords: CELLS ; CELL ; Germany ; PATHWAYS ; QUANTIFICATION ; SYSTEM ; SYSTEMS ; microarray ; PROTEIN ; PROTEINS ; SAMPLES ; DRUG ; BIOLOGY ; SIGNAL ; ASSAY ; microarrays ; EFFICIENT ; ELECTROPHORESIS ; BIOPSY ; PROTEOMICS ; signaling ; RECOMBINANT ; RE ; CAPACITY ; ARRAY ; GELS ; analysis ; methods ; BIOPSIES ; signaling networks ; protein arrays ; protein quantification ; reverse phase protein microarray
    Abstract: The advancement of efficient technologies to comply with the needs of systems biology and drug discovery has so far not received adequate attention. A substantial bottleneck for the time-resolved quantitative description of signaling networks is the limited throughput and the inadequate sensitivity of currently established methods. Here, we present an improved protein microarray-based approach towards the sensitive detection of proteins in the fg-range which is based on signal detection in the near-infrared range. The high sensitivity of the assay permits the specific quantification of proteins derived from as little as only 20 000 cells with an error rate of only 5%. The capacity is limited to the analysis of up to 500 different samples per microarray. Protein abundance is determined qualitatively, and quantitatively, if recombinant protein is available. This novel approach was called IPAQ (infrared-based protein arrays with quantitative readout). IPAQ offers a highly sensitive experimental approach superior to the established standard protein quantification technologies, and is suitable for quantitative proteomics. Employing the IPAQ approach, a detailed analysis of activated signaling networks in biopsy samples and of crosstalk between signaling modules as required in drug discovery strategies can easily be performed
    Type of Publication: Journal article published
    PubMed ID: 17309101
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  • 7
    Keywords: RECEPTOR ; CANCER ; EXPRESSION ; GROWTH ; GROWTH-FACTOR ; CELL ; Germany ; INHIBITION ; KINASE ; PATHWAY ; QUANTIFICATION ; SYSTEM ; SYSTEMS ; DISTINCT ; GENOME ; microarray ; PROTEIN ; PROTEINS ; SAMPLE ; SAMPLES ; ACTIVATION ; BIOLOGY ; MOLECULAR-BIOLOGY ; BREAST ; breast cancer ; BREAST-CANCER ; STIMULATION ; microarrays ; ARRAYS ; systems biology ; PROTEOMICS ; EPIDERMAL-GROWTH-FACTOR ; signaling ; molecular biology ; molecular ; RE ; ARRAY ; EGFR ; analysis ; methods ; HIGH-THROUGHPUT ; HER2 ; GEFITINIB ; comparison ; BREAST-CANCER-CELLS ; EGF ; ERK1/2 ; reverse phase protein array ; HER2/neu ; herceptin
    Abstract: Protein microarrays allow highly accurate comparison and quantification of numerous biological samples in parallel while requiring only little material. This qualifies protein arrays for systems biology and clinical research where only limited sample material is available, but a precise read-out is required. With the introduction of signal normalization steps to monitor the drop size of manually contact-spotted RP protein arrays, the usefulness of normalizer proteins to ensure a high-throughput but inexpensive protein analysis was demonstrated. This approach was applied for the analysis of signaling through ERBB receptor activated kinases in the breast cancer cell line MCF-7. Activation of ERK1/2 and AKT by ERBB1 (EGFR), ERRB2 (HER2/neu), and ERBB3-4 was monitored in a time-resolved manner. Analysis of pathway activation by stimulation with epidermal growth factor and heregulin, or inhibition by blocking with gefitinib or herceptin allowed a characterization of the distinct signaling properties of the different ERBB receptor subtypes
    Type of Publication: Journal article published
    PubMed ID: 18351692
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  • 8
    Keywords: ACTIVATION, analysis, antibodies, antibody, Antibody array, ARRAYS, CAPACITY, CELLS, DESIGN, EXPRESS
    Abstract: A significant bottleneck for the time-resolved and quantitative description of signaling networks is the limited sample capacity and sensitivity of existing methods. Recently, antibody microarrays have emerged as a promising experimental platform for the quantitative and comprehensive determination of protein abundance and protein phosphorylation. This review summarizes the development of microarray applications involving antibody-based capture of target proteins with a focus on quantitative applications. Technical aspects regarding the production of antibody microarrays, identification of suitable detection and capture antibody pairs, signal detection methods, detection limit, and data analysis are discussed in detail
    Type of Publication: Journal article published
    PubMed ID: 18528667
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  • 9
    Abstract: To identify new interactions as well as diagnostically, prognostically and therapeutically relevant differences in the regulation of gene expression in gastrointestinal stromal tumors (GISTs), we analyzed the methylation status, mRNA expression, microRNA expression, protein expression and protein phosphorylation in parallel in identical tumor tissue samples. The data were analyzed in a multilayer approach and were correlated to each other and to clinico-pathological parameters. Differentially regulated genes were mapped to signal transduction pathways which are already known to play a major role in GISTs. A functionally orientated overview of the different data layers was constructed, which enabled new insights into gene regulation in GISTs.
    Type of Publication: Journal article published
    PubMed ID: 20714898
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  • 10
    Keywords: microarrays ; PROTEIN ; microarray ; TOOL ; GENERATION ; reverse phase protein microarray ; USA ; TOOLS ; ARRAY ; analysis ; PHASE
    Type of Publication: Meeting abstract published
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