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Reverse-phase protein array

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https://www.readbyqxmd.com/read/28097235/a-patient-derived-xenograft-platform-to-study-brca-deficient-ovarian-cancers
#1
Erin George, Hyoung Kim, Clemens Krepler, Brandon Wenz, Mehran Makvandi, Janos L Tanyi, Eric Brown, Rugang Zhang, Patricia Brafford, Stephanie Jean, Robert H Mach, Yiling Lu, Gordon B Mills, Meenhard Herlyn, Mark Morgan, Xiaochen Zhang, Robert Soslow, Ronny Drapkin, Neil Johnson, Ying Zheng, George Cotsarelis, Katherine L Nathanson, Fiona Simpkins
Approximately 50% of high-grade serous ovarian cancers (HGSOCs) have defects in genes involved in homologous recombination (HR) (i.e., BRCA1/2). Preclinical models to optimize therapeutic strategies for HR-deficient (HRD) HGSOC are lacking. We developed a preclinical platform for HRD HGSOCs that includes primary tumor cultures, patient-derived xenografts (PDXs), and molecular imaging. Models were characterized by immunohistochemistry, targeted sequencing, and reverse-phase protein array analysis. We also tested PDX tumor response to PARP, CHK1, and ATR inhibitors...
January 12, 2017: JCI Insight
https://www.readbyqxmd.com/read/28092050/microwestern-arrays-for-systems-level-analysis-of-sh2-domain-containing-proteins
#2
Mark F Ciaccio, Richard B Jones
The Microwestern Array (MWA) method combines the scalability and miniaturization afforded by the Reverse Phase Lysate Array (RPLA) approach with the electrophoretic separation characteristic of the Western blot. This technology emulates the creation of an array of small Western blots on a single sheet of nitrocellulose allowing for the sensitive and quantitative measurement of hundreds of proteins from hundreds of cell lysates with minimal cost and maximal accuracy, precision, and reproducibility. The MWA is a versatile technology that can be easily configured for purposes such as antibody screening, cell signaling network inference, protein modification/phenotype regression analysis, and genomic/proteomic relationships...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092031/expression-and-production-of-sh2-domain-proteins
#3
Bernard A Liu, Mari Ogiue-Ikeda, Kazuya Machida
The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP)...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28042144/novel-bet-protein-proteolysis-targeting-chimera-bet-protac-exerts-superior-lethal-activity-than-bromodomain-inhibitor-beti-against-post-myeloproliferative-neoplasm-mpn-secondary-s-aml-cells
#4
D T Saenz, W Fiskus, Y Qian, T Manshouri, K Rajapakshe, K Raina, K G Coleman, A P Crew, A Shen, C P Mill, B Sun, P Qiu, T M Kadia, N Pemmaraju, C DiNardo, M-S Kim, A J Nowak, C Coarfa, C M Crews, S Verstovsek, K N Bhalla
The PROTAC (proteolysis-targeting chimera) ARV-825 recruits bromodomain and extraterminal (BET) proteins to the E3 ubiquitin ligase cereblon, leading to degradation of BET proteins, including BRD4. Whereas the BET-protein inhibitor (BETi) OTX015 caused accumulation of BRD4, treatment with equimolar concentrations of ARV-825 caused sustained and profound depletion (>90%) of BRD4 and induced significantly more apoptosis in cultured and patient-derived (PD) CD34+ post-MPN sAML cells, while relatively sparing the CD34+ normal hematopoietic progenitor cells...
January 2, 2017: Leukemia: Official Journal of the Leukemia Society of America, Leukemia Research Fund, U.K
https://www.readbyqxmd.com/read/28017967/duvelisib-treatment-is-associated-with-altered-expression-of-apoptotic-regulators-that-helps-in-sensitization-of-chronic-lymphocytic-leukemia-cells-to-venetoclax-abt-199
#5
V M Patel, K Balakrishnan, M Douglas, T Tibbitts, E Y Xu, J L Kutok, M Ayers, A Sarkar, R Guerrieri, W G Wierda, S O'Brien, N Jain, H M Stern, V Gandhi
Duvelisib, an oral dual inhibitor of PI3K-δ and PI3K-γ, is in phase III trials for the treatment of chronic lymphocytic leukemia (CLL) and indolent non-Hodgkin's lymphoma (iNHL). In CLL, duvelisib monotherapy is associated with high iwCLL and nodal response rates, but complete remissions are rare. To characterize the molecular effect of duvelisib, we obtained samples from CLL patients on the duvelisib phase I trial. Gene-expression studies (RNA seq, Nanostring, Affymetrix array, and real time RT-PCR) demonstrated increased expression of BCL2 along with several BH3-only pro-apoptotic genes...
December 26, 2016: Leukemia: Official Journal of the Leukemia Society of America, Leukemia Research Fund, U.K
https://www.readbyqxmd.com/read/28017544/context-specificity-in-causal-signaling-networks-revealed-by-phosphoprotein-profiling
#6
Steven M Hill, Nicole K Nesser, Katie Johnson-Camacho, Mara Jeffress, Aimee Johnson, Chris Boniface, Simon E F Spencer, Yiling Lu, Laura M Heiser, Yancey Lawrence, Nupur T Pande, James E Korkola, Joe W Gray, Gordon B Mills, Sach Mukherjee, Paul T Spellman
Signaling networks downstream of receptor tyrosine kinases are among the most extensively studied biological networks, but new approaches are needed to elucidate causal relationships between network components and understand how such relationships are influenced by biological context and disease. Here, we investigate the context specificity of signaling networks within a causal conceptual framework using reverse-phase protein array time-course assays and network analysis approaches. We focus on a well-defined set of signaling proteins profiled under inhibition with five kinase inhibitors in 32 contexts: four breast cancer cell lines (MCF7, UACC812, BT20, and BT549) under eight stimulus conditions...
December 19, 2016: Cell Systems
https://www.readbyqxmd.com/read/27980214/the-isg15-specific-protease-usp18-regulates-stability-of-pten
#7
Lisa Maria Mustachio, Masanori Kawakami, Yun Lu, Jaime Rodriguez-Canales, Barbara Mino, Carmen Behrens, Ignacio Wistuba, Neus Bota-Rabassedas, Jun Yu, J Jack Lee, Jason Roszik, Lin Zheng, Xi Liu, Sarah J Freemantle, Ethan Dmitrovsky
The ubiquitin-like modifier interferon-stimulated gene 15 (ISG15) is implicated in both oncogenic and tumor suppressive programs. Yet, few ISGylation substrates are known and functionally validated in cancer biology. We previously found specific oncoproteins were substrates of ISGylation and were stabilized by the ISG15-specific deubiquitinase (DUB) ubiquitin specific peptidase 18 (USP18). Using reverse-phase protein arrays (RPPAs), this study reports that engineered loss of the DUB USP18 destabilized the tumor suppressor protein phosphatase and tensin homologue (PTEN) in both murine and human lung cancer cell lines...
December 12, 2016: Oncotarget
https://www.readbyqxmd.com/read/27933927/three-dimensionally-functionalized-reverse-phase-glycoprotein-array-for-cancer-biomarker-discovery-and-validation
#8
Li Pan, Hillary Andaluz Aguilar, Linna Wang, Anton Iliuk, W Andy Tao
Glycoproteins have vast structural diversity that plays an important role in many biological processes and have great potential as disease biomarkers. Here, we report a novel functionalized reverse phase protein array (RPPA), termed polymer-based reverse phase glycoprotein array (polyGPA), to capture and profile glycoproteomes specifically, and validate glycoproteins. Nitrocellulose membrane functionalized with globular hydroxyaminodendrimers was used to covalently capture preoxidized glycans on glycoproteins from complex protein samples such as biofluids...
November 30, 2016: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/27883284/novel-biomarkers-of-nasopharyngeal-carcinoma-metastasis-risk-identified-by-reverse-phase-protein-array-based-tumor-profiling-with-consideration-of-plasma-epstein-barr-virus-dna-load
#9
Tao Xu, Bojin Su, Peiyu Huang, Weihong Wei, Yanming Deng, Vasudha Sehgal, Donghui Wang, Jun Jiang, Guoyi Zhang, Anfei Li, Huiling Yang, Francois X Claret
PURPOSE: In patients with Epstein-Barr virus (EBV) associated nasopharyngeal carcinoma (NPC), intertumor heterogeneity causes interpatient heterogeneity in the risk of distant metastasis. We aimed to identify novel biomarkers of metastasis risk using reverse phase protein array (RPPA) profiling of NPC patients at risk for metastasis and considering plasma EBV DNA load. EXPERIMENTAL DESIGN: A total of 98 patients with NPC with and without metastasis after treatment, matched with respect to clinical parameters, are enrolled...
November 24, 2016: Proteomics. Clinical Applications
https://www.readbyqxmd.com/read/27858266/reverse-phase-protein-arrays-enable-glioblastoma-molecular-subtyping
#10
Gregor Hutter, Martin Sailer, Tej Deepak Azad, André O von Bueren, Peter Nollau, Stephan Frank, Cristobal Tostado, Durga Sarvepalli, Arkasubhra Ghosh, Marie-Françoise Ritz, Jean-Louis Boulay, Luigi Mariani
In the present study we investigated the phosphorylation status of the 12 most important signaling cascades in glioblastomas. More than 60 tumor and control biopsies from tumor center and periphery (based on neuronavigation) were subjected to selective protein expression analysis using reverse-phase protein arrays (RPPA) incubated with antibodies against posttranslationally modified cancer pathway proteins. The ratio between phosphorylated (or modified) and non-phosphorylated protein was assessed. All samples were histopathologically validated and proteomic profiles correlated with clinical and survival data...
November 17, 2016: Journal of Neuro-oncology
https://www.readbyqxmd.com/read/27813428/how-may-targeted-proteomics-complement-genomic-data-in-breast-cancer
#11
Mathilde Guerin, Anthony Gonçalves, Yves Toiron, Emilie Baudelet, Stéphane Audebert, Jean-Baptiste Boyer, Jean-Paul Borg, Luc Camoin
Breast cancer (BC) is the most common female cancer in the world and was recently deconstructed in different molecular entities. Although most of the recent assays to characterize tumors at the molecular level are genomic-based, proteins are the actual executors of cellular functions and represent the vast majority of targets for anticancer drugs. Accumulated data has demonstrated an important level of quantitative and qualitative discrepancies between genomic/transcriptomic alterations and their protein counterparts, mostly related to the large number of post-translational modifications...
January 2017: Expert Review of Proteomics
https://www.readbyqxmd.com/read/27771839/transcriptome-and-proteome-oriented-identification-of-dysregulated-eif4g-stat3-and-hippo-pathways-altered-by-pik3ca-h1047r-in-her2-er-positive-breast-cancer
#12
Feixiong Cheng, Junfei Zhao, Ariella B Hanker, Monica Red Brewer, Carlos L Arteaga, Zhongming Zhao
PURPOSE: Phosphatidylinositol 3-kinase (PI3K)/AKT pathway aberrations are common in human breast cancer. Furthermore, PIK3CA mutations are commonly associated with resistance to anti-epidermal growth factor receptor 2 (HER2) or anti-estrogen receptor (ER) agents in HER2 or ER positive (HER2(+)/ER(+)) breast cancer. Hence, deciphering the underlying mechanisms of PIK3CA mutations in HER2(+)/ER(+) breast cancer would provide novel insights into elucidating resistance to anti-HER2/ER therapies...
October 22, 2016: Breast Cancer Research and Treatment
https://www.readbyqxmd.com/read/27764801/foxm1-and-stat3-interaction-confers-radioresistance-in-glioblastoma-cells
#13
Uday B Maachani, Uma Shankavaram, Tamalee Kramp, Philip J Tofilon, Kevin Camphausen, Anita T Tandle
Glioblastoma multiforme (GBM) continues to be the most frequently diagnosed and lethal primary brain tumor. Adjuvant chemo-radiotherapy remains the standard of care following surgical resection. In this study, using reverse phase protein arrays (RPPAs), we assessed the biological effects of radiation on signaling pathways to identify potential radiosensitizing molecular targets. We identified subsets of proteins with clearly concordant/discordant behavior between irradiated and non-irradiated GBM cells in vitro and in vivo...
October 14, 2016: Oncotarget
https://www.readbyqxmd.com/read/27754870/transgenic-expression-of-the-mitochondrial-chaperone-tnfr-associated-protein-1-trap1-accelerates-prostate-cancer-development
#14
Sofia Lisanti, David S Garlick, Kelly G Bryant, Michele Tavecchio, Gordon B Mills, Yiling Lu, Andrew V Kossenkov, Louise C Showe, Lucia R Languino, Dario C Altieri
Protein homeostasis, or proteostasis, is required for mitochondrial function, but its role in cancer is controversial. Here we show that transgenic mice expressing the mitochondrial chaperone TNFR-associated protein 1 (TRAP1) in the prostate develop epithelial hyperplasia and cellular atypia. When examined on a Pten(+/-) background, a common alteration in human prostate cancer, TRAP1 transgenic mice showed accelerated incidence of invasive prostatic adenocarcinoma, characterized by increased cell proliferation and reduced apoptosis, in situ Conversely, homozygous deletion of TRAP1 delays prostatic tumorigenesis in Pten(+/-) mice without affecting hyperplasia or prostatic intraepithelial neoplasia...
November 25, 2016: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/27751915/defining-the-na-h-exchanger-nhe1-interactome-in-triple-negative-breast-cancer-cells
#15
Schammim Ray Amith, Krista Marie Vincent, Jodi Marie Wilkinson, Lynne Marie Postovit, Larry Fliegel
Mounting evidence supports a major role for the Na(+)/H(+) exchanger NHE1 in cancer progression and metastasis. NHE1 is hyperactive at the onset of oncogenic transformation, resulting in intracellular alkalinization and extracellular microenvironmental acidification. These conditions promote invasion and facilitate metastasis. However, the signal pathways governing the regulation of exchanger activity are still unclear. This is especially important in the aggressively metastatic, triple-negative basal breast cancer subtype...
January 2017: Cellular Signalling
https://www.readbyqxmd.com/read/27677740/bet-protein-bromodomain-inhibitor-based-combinations-are-highly-active-against-post-myeloproliferative-neoplasm-secondary-aml-cells
#16
D T Saenz, W Fiskus, T Manshouri, K Rajapakshe, S Krieger, B Sun, C P Mill, C DiNardo, N Pemmaraju, T Kadia, S Parmar, S Sharma, C Coarfa, P Qiu, S Verstovsek, K N Bhalla
Myeloproliferative neoplasms with myelofibrosis (MPN-MF) demonstrate constitutive activation of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling that responds to treatment with the JAK1 and 2 kinase inhibitor (JAKi) ruxolitinib. However, MPN-MF often progresses (~20%) to secondary acute myeloid leukemia (sAML), where standard induction chemotherapy or ruxolitinib is relatively ineffective, necessitating the development of novel therapeutic approaches. In the present studies, we demonstrate that treatment with BET (bromodomain and extraterminal) protein inhibitor (BETi), for example, JQ1, inhibits growth and induces apoptosis of cultured and primary, patient-derived (PD), post-MPN sAML blast progenitor cells...
October 25, 2016: Leukemia: Official Journal of the Leukemia Society of America, Leukemia Research Fund, U.K
https://www.readbyqxmd.com/read/27663479/using-reverse-phase-protein-arrays-as-pharmacodynamic-assays-for-functional-proteomics-biomarker-discovery-and-drug-development-in-cancer
#17
REVIEW
Yiling Lu, Shiyun Ling, Apurva M Hegde, Lauren A Byers, Kevin Coombes, Gordon B Mills, Rehan Akbani
The majority of the targeted therapeutic agents in clinical use target proteins and protein function. Although DNA and RNA analyses have been used extensively to identify novel targets and patients likely to benefit from targeted therapies, these are indirect measures of the levels and functions of most therapeutic targets. More importantly, DNA and RNA analysis is ill-suited for determining the pharmacodynamic effects of target inhibition. Assessing changes in protein levels and function is the most efficient way to evaluate the mechanisms underlying sensitivity and resistance to targeted agents...
August 2016: Seminars in Oncology
https://www.readbyqxmd.com/read/27658583/hypoxia-promotes-chemoresistance-in-acute-lymphoblastic-leukemia-cell-lines-by-modulating-death-signaling-pathways
#18
C Petit, F Gouel, I Dubus, C Heuclin, K Roget, J P Vannier
BACKGROUND: Several studies show that bone marrow (BM) microenvironment and hypoxia condition can promote the survival of leukemic cells and induce resistance to anti-leukemic drugs. However, the molecular mechanism for chemoresistance by hypoxia is not fully understood. METHODS: In the present study, we investigated the effect of hypoxia on resistance to two therapies, methotrexate (MTX) and prednisolone (PRD), in two cell models for acute lymphoblastic leukemia (ALL)...
2016: BMC Cancer
https://www.readbyqxmd.com/read/27622568/reverse-phase-protein-arrays-for-compound-profiling
#19
Nathan Moerke, Mohammad Fallahi-Sichani
Reverse phase protein arrays (RPPAs), also called reverse phase lysate arrays (RPLAs), involve immobilizing cell or tissue lysates, in small spots, onto solid supports which are then probed with primary antibodies specific for proteins or post-translational modifications of interest. RPPA assays are well suited for large-scale, high-throughput measurement of protein and PTM levels in cells and tissues. RPPAs are affordable and highly multiplexable, as a large number of arrays can readily be produced in parallel and then probed separately with distinct primary antibodies...
2016: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/27612029/atm-chk-p53-pathway-dependent-chemopreventive-and-therapeutic-activity-on-lung-cancer-by-pterostilbene
#20
Hani Lee, Yonghwan Kim, Ji Hye Jeong, Jae-Ha Ryu, Woo-Young Kim
Among the many stilbenoids found in a variety of berries, resveratrol and pterostilbene are of particular interest given their potential for use in cancer therapeutics and prevention. We purified four stilbenoids from R. undulatum and found that pterostilbene inhibits cancer cell proliferation more efficiently than rhapontigenin, piceatannol and resveratrol. To investigate the underlying mechanism of this superior action of pterostilbene on cancer cells, we utilized a reverse-phase protein array followed by bioinformatic analysis and found that the ATM/CHK pathway is modified by pterostilbene in a lung cancer cell line...
2016: PloS One
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