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Search Results: 1 - 10 of 27773 matches for " Ming-Sound Tsao "
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Modified gateway system for double shRNA expression and Cre/lox based gene expression
Nikolina Radulovich, Lisa Leung, Ming-Sound Tsao
BMC Biotechnology , 2011, DOI: 10.1186/1472-6750-11-24
Abstract: Aiming to provide tools for high throughput analysis of gene functions, we have developed a modified short hairpin RNA (shRNA) and gene expression system based on Gateway Technology. The system contains a series of entry and destination vectors that enables easy transfer of shRNA or cDNA into lentiviral expression systems with a variety of selection or marker genes (i.e. puromycin, hygromycin, green fluorescent protein-EGFP, yellow fluorescent protein-YFP and red fluorescent protein-dsRed2). Our shRNA entry vector pENTR.hU6.hH1 containing two tandem human shRNA expression promoters, H1 and U6, was capable of co-expressing two shRNA sequences simultaneously. The entry vector for gene overexpression, pENTR.CMV.ON was constructed to contain CMV promoter with a multiple cloning site flanked by loxP sites allowing for subsequent Cre/lox recombination. Both shRNA and cDNA expression vectors also contained attL sites necessary for recombination with attR sites in our destination expression vectors. As proof of principle we demonstrate the functionality and efficiency of this system by testing expression of several cDNA and shRNA sequences in a number of cell lines.Our system is a valuable addition to already existing library of Gateway based vectors and can be an essential tool for many aspects of gene functional studies.In the era of whole genome sequencing and proteomics, there has never been a greater need to develop versatile tools for gene functional studies. Such studies necessitate a series of genetic manipulations including overexpression and/or downregulation of genes of interest either in in vivo and/or in vitro settings. Downregulation of genes has been made possible by RNA interference (RNAi) technology [1] and targeting genes using expressed short hairpin RNA [2,3] is currently the method of choice by a majority of researchers. The most successful RNAi libraries based on retroviral [4] and lentiviral expression [5,6] of shRNAs have also been utilized for large
Differential utilization of NF-kappaB RELA and RELB in response to extracellular versus intracellular polyIC stimulation in HT1080 cells
James J Yun, Ming-Sound Tsao, Sandy D Der
BMC Immunology , 2011, DOI: 10.1186/1471-2172-12-15
Abstract: Here, we studied an intracellular dsRNA pathway in the human fibrosarcoma cell line HT1080, which is distinct from the TLR3-mediated extracellular dsRNA pathway. Particularly, the NF-kB subunits RELA and RELB were differentially utilized by these two dsRNA signaling pathways. In TLR3-mediated dsRNA signaling, siRNA knock-down studies suggested a limited role for RELA on regulation of interferon beta and other cytokines whereas RELB appeared to have a negative regulatory role. By contrast, intracellular dsRNA signaling was dependent on RELA, but not RELB.Our study suggests that extracellular and intracellular dsRNA signaling pathways may utilize different NF-kB members, and particularly the differential utilization of RELB may be a key mechanism for powerful inductions of NF-kB regulated genes in the intracellular dsRNA signaling pathway.Pattern recognition receptors (PRRs) are key players in host innate immune response against microbial pathogens. In order to launch effective defense mechanisms in response to viral infections, a number of cellular sensors that recognize universal components common to many viruses have been characterized. Double-stranded RNA (dsRNA) is one of the components that mammalian cells have developed several different receptors for since most viruses produce dsRNA during replication [1-3].Interferon-inducible double-stranded RNA activated protein kinase (PKR) has long been studied as an intracellular sensor for viral dsRNA. PKR was initially characterized to participate in the mechanism that shuts down cellular translation to suppress viral replication and is now believed to be involved in a wide range of other cellular responses to viral infection [4]. Toll-like receptor 3 (TLR3) has been considered to be essential for mediating NF-kB-inducible gene responses to polyIC, a synthetic analogue of viral dsRNA [5], but there has yet been any strong evidence of physical interaction between TLR3 and viral dsRNA. The precise cellular location of TL
Ancillary Testing in Lung Cancer Diagnosis
William Dubinski,Natasha B. Leighl,Ming-Sound Tsao,David M. Hwang
Pulmonary Medicine , 2012, DOI: 10.1155/2012/249082
Abstract: The pathologic diagnosis of lung cancer historically has relied primarily on morphologic features of tumors in histologic sections. With the emergence of new targeted therapies, the pathologist is called upon increasingly to provide not only accurate typing of lung cancers, but also to provide prognostic and predictive information, based on a growing number of ancillary tests, that may have significant impact on patient management. This review provides an overview of ancillary tests currently used in the pathologic diagnosis of lung cancer, with a focus on immunohistochemistry and molecular diagnostics. 1. Introduction Primary lung cancer has been classified historically into two clinically relevant groups: small cell lung cancer (SCLC) and nonsmall cell lung cancer (NSCLC). This distinction was clinically useful as available treatment strategies differed significantly between these two groups. In recent years, the emerging evidence of differential response to new targeted therapies and the identification of molecular differences between specific subtypes of NSCLC increasingly necessitate greater accuracy in the subtyping of NSCLC. The current WHO classification of lung cancer [1] has been based almost entirely by assessment of morphologic features using standard hematoxylin and eosin (H&E) stained sections of tumors. However, a growing number of ancillary studies can help with classification, such as the use of immunohistochemistry (IHC). Beyond simple classification, however, ancillary testing for molecular aberrations is entering routine practice and delivers additional prognostic and predictive information. A new multidisciplinary classification system for primary lung adenocarcinomas has emerged recently [2]. While this system is still based largely on morphology, it moves towards incorporating recent advances in clinical and molecular medicine. In this review, we summarize ancillary tests currently used in the pathologic diagnosis of lung cancer, with a focus on immunohistochemistry and molecular diagnostics. 2. Immunohistochemistry Immunohistochemistry involves the detection and localization of antigens or proteins in tissue sections by the use of antibodies that bind specifically to the antigen of interest. The antibodies are coupled to a detection system which allows them to be visualized in tissue sections. IHC has a range of applications in the practice of pathology and is commonly used by pathologists to help in distinguishing cell types or their origin, using markers that are expressed differentially between different cell types and organs.
Immunohistochemical analysis of changes in signaling pathway activation downstream of growth factor receptors in pancreatic duct cell carcinogenesis
Nhu-An Pham, Joerg Schwock, Vladimir Iakovlev, Greg Pond, David W Hedley, Ming-Sound Tsao
BMC Cancer , 2008, DOI: 10.1186/1471-2407-8-43
Abstract: We performed immunohistochemistry on a tissue microarray of 26 PDAC, 13 normal appearing adjacent pancreatic ductal epithelia, and 12 normal non-PDAC ducts. We compared the levels of 18 signaling proteins including growth factor receptors, tumor suppressors and 13 of their putative downstream phosphorylated (p-) signal transducers in PDAC to those in normal ductal epithelia.The overall profiles of signaling protein expression levels, activation states and sub-cellular distribution in PDAC cells were distinguishable from non-neoplastic ductal epithelia. The ERK pathway activation was correlated with high levels of S2448p-mTOR (100%, p = 0.05), T389p-S6K (100%, p = 0.02 and S235/236p-S6 (86%, p = 0.005). Additionally, T389p-S6K correlated with S727p-STAT3 (86%, p = 0.005). Advanced tumors with lymph node metastasis were characterized by high levels of S276p-NFκB (100%, p = 0.05) and S9p-GSK3β (100%, p = 0.05). High levels of PKBβ/AKT2, EGFR, as well as nuclear T202/Y204p-ERK and T180/Y182p-p38 were observed in normal ducts adjacent to PDAC compared with non-cancerous pancreas.Multiple signaling proteins are activated in pancreatic duct cell carcinogenesis including those associated with the ERK, PKB/AKT, mTOR and STAT3 pathways. The ERK pathway activation appears also increased in duct epithelia adjacent to carcinoma, suggesting tumor micro-environmental effects.Pancreatic ductal adenocarcinoma (PDAC) is the most common malignant tumor of the human pancreas. PDAC patients have one of the worst prognoses among all cancer types with a 5-year survival rate of less than 5%. Despite significant advances during the last decade in our molecular knowledge on this disease, the prognosis and management of PDAC patients have remained unchanged [1,2]. The most common genetic aberrations in pancreatic duct cell carcinogenesis involve the activation of KRAS oncogene and inactivation of tumor suppressor genes p16/CDKN2, p53 and SMAD4/DPC4 [3]. Less frequently altered genes in PDAC a
Differential roles of cyclin D1 and D3 in pancreatic ductal adenocarcinoma
Nikolina Radulovich, Nhu-An Pham, Dan Strumpf, Lisa Leung, Wing Xie, Igor Jurisica, Ming-Sound Tsao
Molecular Cancer , 2010, DOI: 10.1186/1476-4598-9-24
Abstract: CCND1 and CCND3 expression were selectively suppressed by shRNA in PDAC cell lines with expression levels of equal CCND1 and CCND3 (BxPC3), enhanced CCND1 (HPAC) or enhanced CCND3 (PANC1). Suppression of cell proliferation was greater with CCND3 than CCND1 downregulation. CCND3 suppression led to a reduced level of phosphorylated retinoblastoma protein (Ser795p-Rb/p110) and resulted in decreased levels of cyclin A mRNA and protein. A global gene expression analysis identified deregulated genes in D1- or D3-cyclin siRNA-treated PANC1 cells. The downregulated gene targets in CCND3 suppressed cells were significantly enriched in cell cycle associated processes (p < 0.005). In contrast, focal adhesion/actin cytoskeleton, MAPK and NF B signaling appeared to characterize the target genes and their interacting proteins in CCND1 suppressed PANC1 cells.Our results suggest that CCND3 is the primary driver of the cell cycle, in cooperation with CCND1 that integrates extracellular mitogenic signaling. We also present evidence that CCND1 plays a role in tumor cell migration. The results provide novel insights for common and differential targets of CCND1 and CCND3 overexpression during pancreatic duct cell carcinogenesis.In normal cells, growth factors and mitogenic signaling stimulate the expression of D-cyclins and E2F activity to drive G0/G1 to S phase cell cycle progression [1]. D-cyclins bind to and activate CDK4/6, which phosphorylate the retinoblastoma tumor suppressor protein (Rb) leading to its inactivation and the release of the E2F transcription factors and expression of genes critical for cell cycle progression. In many human cancers, one or more of these regulators for G1/S cell cycle transition are often altered in their expression or function [2]. The inactivation of the tumor suppressor p16 [3] and the overexpression of cyclin D1 (CCND1) and/or cyclin D3 (CCND3) are common in pancreatic ductal adenocarcinoma (PDAC). During multi-stage pancreatic duct cell carcinog
Loss of Canonical Smad4 Signaling Promotes KRAS Driven Malignant Transformation of Human Pancreatic Duct Epithelial Cells and Metastasis
Lisa Leung, Nikolina Radulovich, Chang-Qi Zhu, Dennis Wang, Christine To, Emin Ibrahimov, Ming-Sound Tsao
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0084366
Abstract: Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer death in North America. Activating KRAS mutations and Smad4 loss occur in approximately 90% and 55% of PDAC, respectively. While their roles in the early stages of PDAC development have been confirmed in genetically modified mouse models, their roles in the multistep malignant transformation of human pancreatic duct cells have not been directly demonstrated. Here, we report that Smad4 represents a barrier in KRAS-mediated malignant transformation of the near normal immortalized human pancreatic duct epithelial (HPDE) cell line model. Marked Smad4 downregulation by shRNA in KRASG12V expressing HPDE cells failed to cause tumorigenic transformation. However, KRAS-mediated malignant transformation occurred in a new HPDE-TGF-β resistant (TβR) cell line that completely lacks Smad4 protein expression and is resistant to the mito-inhibitory activity of TGF-β. This transformation resulted in tumor formation and development of metastatic phenotype when the cells were implanted orthotopically into the mouse pancreas. Smad4 restoration re-established TGF-β sensitivity, markedly increased tumor latency by promoting apoptosis, and decreased metastatic potential. These results directly establish the critical combination of the KRAS oncogene and complete Smad4 inactivation in the multi-stage malignant transformation and metastatic progression of normal human HPDE cells.
Transcriptome Profiles of Carcinoma-in-Situ and Invasive Non-Small Cell Lung Cancer as Revealed by SAGE
Kim M. Lonergan,Raj Chari,Bradley P. Coe,Ian M. Wilson,Ming-Sound Tsao,Raymond T. Ng,Calum MacAulay,Stephen Lam,Wan L. Lam
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0009162
Abstract: Non-small cell lung cancer (NSCLC) presents as a progressive disease spanning precancerous, preinvasive, locally invasive, and metastatic lesions. Identification of biological pathways reflective of these progressive stages, and aberrantly expressed genes associated with these pathways, would conceivably enhance therapeutic approaches to this devastating disease.
Tumor Cell Marker PVRL4 (Nectin 4) Is an Epithelial Cell Receptor for Measles Virus
Ryan S. Noyce,Daniel G. Bondre,Michael N. Ha,Liang-Tzung Lin,Gary Sisson,Ming-Sound Tsao,Christopher D. Richardson
PLOS Pathogens , 2011, DOI: 10.1371/journal.ppat.1002240
Abstract: Vaccine and laboratory adapted strains of measles virus can use CD46 as a receptor to infect many human cell lines. However, wild type isolates of measles virus cannot use CD46, and they infect activated lymphocytes, dendritic cells, and macrophages via the receptor CD150/SLAM. Wild type virus can also infect epithelial cells of the respiratory tract through an unidentified receptor. We demonstrate that wild type measles virus infects primary airway epithelial cells grown in fetal calf serum and many adenocarcinoma cell lines of the lung, breast, and colon. Transfection of non-infectable adenocarcinoma cell lines with an expression vector encoding CD150/SLAM rendered them susceptible to measles virus, indicating that they were virus replication competent, but lacked a receptor for virus attachment and entry. Microarray analysis of susceptible versus non-susceptible cell lines was performed, and comparison of membrane protein gene transcripts produced a list of 11 candidate receptors. Of these, only the human tumor cell marker PVRL4 (Nectin 4) rendered cells amenable to measles virus infections. Flow cytometry confirmed that PVRL4 is highly expressed on the surfaces of susceptible lung, breast, and colon adenocarcinoma cell lines. Measles virus preferentially infected adenocarcinoma cell lines from the apical surface, although basolateral infection was observed with reduced kinetics. Confocal immune fluorescence microscopy and surface biotinylation experiments revealed that PVRL4 was expressed on both the apical and basolateral surfaces of these cell lines. Antibodies and siRNA directed against PVRL4 were able to block measles virus infections in MCF7 and NCI-H358 cancer cells. A virus binding assay indicated that PVRL4 was a bona fide receptor that supported virus attachment to the host cell. Several strains of measles virus were also shown to use PVRL4 as a receptor. Measles virus infection reduced PVRL4 surface expression in MCF7 cells, a property that is characteristic of receptor-associated viral infections.
Exploiting the noise: improving biomarkers with ensembles of data analysis methodologies
Maud HW Starmans, Melania Pintilie, Thomas John, Sandy D Der, Frances A Shepherd, Igor Jurisica, Philippe Lambin, Ming-Sound Tsao, Paul C Boutros
Genome Medicine , 2012, DOI: 10.1186/gm385
Abstract: We evaluated two published prognostic multi-gene biomarkers for NSCLC in an independent 442-patient dataset. We then systematically assessed how technical factors influenced validation success.Both biomarkers validated successfully (biomarker #1: hazard ratio (HR) 1.63, 95% confidence interval (CI) 1.21 to 2.19, P = 0.001; biomarker #2: HR 1.42, 95% CI 1.03 to 1.96, P = 0.030). Further, despite being underpowered for stage-specific analyses, both biomarkers successfully stratified stage II patients and biomarker #1 also stratified stage IB patients. We then systematically evaluated reasons for reported validation failures and find they can be directly attributed to technical challenges in data analysis. By examining 24 separate pre-processing techniques we show that minor alterations in pre-processing can change a successful prognostic biomarker (HR 1.85, 95% CI 1.37 to 2.50, P < 0.001) into one indistinguishable from random chance (HR 1.15, 95% CI 0.86 to 1.54, P = 0.348). Finally, we develop a new method, based on ensembles of analysis methodologies, to exploit this technical variability to improve biomarker robustness and to provide an independent confidence metric.Biomarkers comprise a fundamental component of personalized medicine. We first validated two NSCLC prognostic biomarkers in an independent patient cohort. Power analyses demonstrate that even this large, 442-patient cohort is under-powered for stage-specific analyses. We then use these results to discover an unexpected sensitivity of validation to subtle data analysis decisions. Finally, we develop a novel algorithmic approach to exploit this sensitivity to improve biomarker robustness.The perfect medical treatment would start with instantaneous diagnosis, proceed rapidly to a treatment that offered complete cure with no side effects, and of course would incur minimal costs to the healthcare system. This dream scenario, while distant, has been made more plausible by a rapid reduction in the cost of mol
Quantitative image analysis of immunohistochemical stains using a CMYK color model
Nhu-An Pham, Andrew Morrison, Joerg Schwock, Sarit Aviel-Ronen, Vladimir Iakovlev, Ming-Sound Tsao, James Ho, David W Hedley
Diagnostic Pathology , 2007, DOI: 10.1186/1746-1596-2-8
Abstract: We adapted a Cyan/Magenta/Yellow/Key (CMYK) model for automated computer image analysis to quantify IHC stains in hematoxylin counterstained histological sections.The spectral characteristics of the chromogens AEC, DAB and NovaRed as well as the counterstain hematoxylin were first determined using CMYK, Red/Green/Blue (RGB), normalized RGB and Hue/Saturation/Lightness (HSL) color models. The contrast of chromogen intensities on a 0–255 scale (24-bit image file) as well as compared to the hematoxylin counterstain was greatest using the Yellow channel of a CMYK color model, suggesting an improved sensitivity for IHC evaluation compared to other color models. An increase in activated STAT3 levels due to growth factor stimulation, quantified using the Yellow channel image analysis was associated with an increase detected by Western blotting. Two clinical image data sets were used to compare the Yellow channel automated method with observer-dependent methods. First, a quantification of DAB-labeled carbonic anhydrase IX hypoxia marker in 414 sections obtained from 138 biopsies of cervical carcinoma showed strong association between Yellow channel and positive color selection results. Second, a linear relationship was also demonstrated between Yellow intensity and visual scoring for NovaRed-labeled epidermal growth factor receptor in 256 non-small cell lung cancer biopsies.The Yellow channel image analysis method based on a CMYK color model is independent of observer biases for threshold and positive color selection, applicable to different chromogens, tolerant of hematoxylin, sensitive to small changes in IHC intensity and is applicable to simple automation procedures. These characteristics are advantageous for both basic as well as clinical research in an unbiased, reproducible and high throughput evaluation of IHC intensity.Immunohistochemistry (IHC) for the evaluation of antigen expression as well as higher resolution methodologies for cytogenetic analysis are standard
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