Springer protocols feed by Cancer Research 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Methods to Identify and Validate WT1–RNA Interaction
Tumor suppressor protein, Wt1 is a transcription factor that binds to DNA sequence similar to the Early Growth Response gene, EGR1 consensus binding sequence. Biophysical and biochemical validations have shown that the zinc fingers of Wt1 are capable of binding to both DNA and RNA albeit with different binding affinities which potentially is also isoform specific. SELEX based identification of the RNA binding motifs led to the identification of motifs which could not be translated into the in vivo context. With the advent of recent technologies that allow cross-linking of RNA and protein and high throughput sequencing tech...
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news
WT1-Associated Protein–Protein Interaction Networks
Tumor-suppressor protein Wt1 has been shown to interact with specific proteins that influence its function. These protein interactions have been identified as direct individual interactions but with the potential to exist as a part of a multiprotein complex. In order to obtain the global proteome interaction map of Wt1, an unbiased label-free endogenous immunoprecipitation was performed followed by mass spectrometry to identify protein interactions that are Wt1 centric. This chapter details the different techniques that have been used to identify and characterize Wt1-interacting proteins. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news
Identifying Direct Downstream Targets: WT1 ChIP-Seq Analysis
Identifying targets of transcriptional regulators such as the Wilms’ tumor-suppressor protein (WT1) is an integral part of understanding the mechanisms governing the spatial and temporal activation of different genes. A commonly used strategy for studying transcription factors involves performing chromatin immunoprecipitation (ChIP) for the protein of interest with an appropriate antibody in crosslinked cells. Following ChIP, the enriched DNA is sequenced using next-generation sequencing (NGS) technologies and the transcription factor target sites are identified via bioinformatics analysis. Here we provide a detailed...
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news
Measuring Equilibrium Binding Constants for the WT1-DNA Interaction Using a Filter Binding Assay
Equilibrium binding of WT1 to specific sites in DNA and potentially RNA molecules is central in mediating the regulatory roles of this protein. In order to understand the functional effects of mutations in the nucleic acid-binding domain of WT1 proteins and/or mutations in the DNA- or RNA-binding sites, it is necessary to measure the equilibrium constant for formation of the protein-nucleic acid complex. This chapter describes the use of a filter binding assay to make accurate measurements of the binding of the WT1 zinc finger domain to the consensus WT1-binding site in DNA. The method described is readily adapted to the m...
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news
In Vitro Transcription to Study WT1 Function
In vitro transcription methods using mammalian nuclear extracts have been available for over 30 years and have allowed sophisticated biochemical analyses of the transcription process. This method has been extensively used to study the basic mechanisms of transcription, allowing the identification of the general transcription factors and elucidation of their mechanisms of action. Gene-specific transcriptional regulators have also been studied using in vitro transcription. This has facilitated the identification of their cofactors and provided information on their function that is invaluable to facilitate their study in a mo...
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news
Fluorescence-Activated Cell Sorting (FACS) Protocol for Podocyte Isolation in Adult Zebrafish
Zebrafish is becoming a very important model for studying human diseases. The conserved structure of the nephrons in the kidney allows the user to answer questions relating to study human kidney disorders. Wt1a-expressing podocytes are the most important cells within the glomeruli of adult zebrafish. In order to understand the molecular characteristics of these cells, within damage models, we have established a method for isolating them. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news
Immunofluorescence Staining of Wt1 on Sections of Zebrafish Embryos and Larvae
Immunohistochemistry is one of the most powerful tools for direct visualization of distribution and localization of gene products. The presented protocol provides an opportunity to determine the localization patterns of Wt1 in zebrafish via antibody staining. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news
Function and Regulation of the Wilms’ Tumor Suppressor 1 (WT1) Gene in Fish
The Wilms’ tumor suppressor gene Wt1 is highly conserved among vertebrates. In contrast to mammals, most fish species possess two wt1 paralogs that have been named wt1a and wt1b. Concerning wt1 in fish, most work so far has been done using zebrafish, focusing on the embryonic kidney, the pronephros. In this chapter we will describe the structure and development of the pronephros as well as the role that the wt1 genes play in the embryonic zebrafish kidney. We also discuss Wt1 target genes and describe the potential function of the Wt1 proteins in the adult kidney. Finally we will summarize data on the role of Wt1 out...
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news
Erratum to: South African Herbal Extracts as Potential Chemopreventive Agents: Screening for Anticancer Splicing Activity
(Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - December 15, 2015 Category: Cancer & Oncology Source Type: news
AlgiMatrix & trade;-Based 3D Cell Culture System as an In Vitro Tumor Model: An Important Tool in Cancer Research
Routinely used two-dimensional cell culture-based models often fail while translating the observations into in vivo models. This setback is more common in cancer research, due to several reasons. The extracellular matrix and cell-to-cell interactions are not present in two-dimensional (2D) cell culture models. Diffusion of drug molecules into cancer cells is hindered by barriers of extracellular components in in vivo conditions, these barriers are absent in 2D cell culture models. To better mimic or simulate the in vivo conditions present in tumors, the current study used the alginate based three-dimensional cell culture (...
Source: Springer protocols feed by Cancer Research - December 14, 2015 Category: Cancer & Oncology Source Type: news
Comet Assay in Cancer Chemoprevention
The comet assay can be useful in monitoring DNA damage in single cells caused by exposure to genotoxic agents, such as those causing air, water, and soil pollution (e.g., pesticides, dioxins, electromagnetic fields) and chemo- and radiotherapy in cancer patients, or in the assessment of genoprotective effects of chemopreventive molecules. Therefore, it has particular importance in the fields of pharmacology and toxicology, and in both environmental and human biomonitoring. It allows the detection of single strand breaks as well as double-strand breaks and can be used in both normal and cancer cells. Here we describe the al...
Source: Springer protocols feed by Cancer Research - November 28, 2015 Category: Cancer & Oncology Source Type: news
1H NMR Metabolomic Footprinting Analysis for the In Vitro Screening of Potential Chemopreventive Agents
Metabolomics is the quantification and analysis of the concentration profiles of low-molecular-weight compounds present in biological samples. In particular metabolic footprinting analysis, based on the monitoring of metabolites consumed from and secreted into the growth medium, is a valuable tool for the study of pharmacological and toxicological effects of drugs. Mass spectrometry and nuclear magnetic resonance (NMR) are the two main complementary techniques used in this field. Although less sensitive, NMR gives a direct fingerprint of the system, and the spectra obtained contain metabolic information that can be distill...
Source: Springer protocols feed by Cancer Research - November 28, 2015 Category: Cancer & Oncology Source Type: news
LC-MS-Based Metabolomic Investigation of Chemopreventive Phytochemical-Elicited Metabolic Events
Phytochemicals are under intensive investigation for their potential use as chemopreventive agents in blocking or suppressing carcinogenesis. Metabolic interactions between phytochemical and biological system play an important role in determining the efficacy and toxicity of chemopreventive phytochemicals. However, complexities of phytochemical biotransformation and intermediary metabolism pose challenges for studying phytochemical-elicited metabolic events. Metabolomics has become a highly effective technical platform to detect subtle changes in a complex metabolic system. Here, using green tea polyphenols as an example, ...
Source: Springer protocols feed by Cancer Research - November 28, 2015 Category: Cancer & Oncology Source Type: news
The Methylated DNA Immunoprecipitation [MeDIP] to Investigate the Epigenetic Remodeling in Cell Fate Determination and Cancer Development
Epigenetic mechanisms such as DNA methylation, posttranslational modifications of histone proteins, remodeling of nucleosomes, and the expression of noncoding RNAs contribute to the regulation of gene expression for the cell fate determination and tissue development. The disruption of these epigenetic mechanisms, in conjunction with genetic alterations, is a decisive element for cancer development and progression. The cancer phenotype is characterized by global DNA hypomethylation and gene-specific hypermethylation. The methylated DNA immunoprecipitation [MeDIP] is a useful approach currently used to clarify the functional...
Source: Springer protocols feed by Cancer Research - November 28, 2015 Category: Cancer & Oncology Source Type: news
Detection of Circulating Tumor DNA in the Blood of Cancer Patients: An Important Tool in Cancer Chemoprevention
Liquid biopsies represent novel promising tools to determine the impact of clonal heterogeneity on clinical outcomes with the potential to identify novel therapeutic targets in cancer patients. We developed a low-coverage whole-genome sequencing approach in order to noninvasively establish copy number aberrations in plasma DNA from metastasized cancer patients. Using plasma-Seq we were able to monitor genetic evolution including the acquirement of novel copy number changes, such as focal amplifications and chromosomal polysomies. The big advantage of our approach is that it can be performed on a benchtop sequencer, speed, ...
Source: Springer protocols feed by Cancer Research - November 28, 2015 Category: Cancer & Oncology Source Type: news
