In Vivo Assays for Assessing the Role of the Wilms & rsquo; Tumor Suppressor 1 (Wt1) in Angiogenesis
The Wilms & rsquo; tumor suppressor gene (WT1) is widely expressed during neovascularization, but it is almost entirely absent in quiescent adult vasculature. However, in vessels undergoing angiogenesis, WT1 is dramatically upregulated. Studies have shown Wt1 has a role in both tumor and ischemic angiogenesis, but the mechanism of Wt1 action in angiogenic tissue remains to be elucidated. Here, we describe two methods for induction of in vivo angiogenesis (subcutaneous sponge implantation, femoral artery ligation) that can be used to assess the influence of Wt1 on new blood vessel formation. Subcutaneously implanted spo...
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

Methods to Identify and Validate WT1 & ndash;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 & ndash;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

Function and Regulation of the Wilms & rsquo; Tumor Suppressor 1 (WT1) Gene in Fish
The Wilms & rsquo; 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 W...
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

Multiphoton Microscopy for Visualizing Lipids in Tissue
Visualizing the appearance of fat droplets and adipocytes in tissue can be realized using a label-free imaging method known as coherent anti-Stokes Raman spectroscopy (CARS). CARS is a nonlinear optical technique that allows label-free imaging of a material with contrast based on the same vibrational signatures of molecules found in Raman spectroscopy. CARS can be combined with other single and multiphoton imaging modes such as second harmonic generation and two-photon fluorescence to image a broad variety of biological structures. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

In Vivo Assays for Assessing the Role of the Wilms’ Tumor Suppressor 1 (Wt1) in Angiogenesis
The Wilms’ tumor suppressor gene (WT1) is widely expressed during neovascularization, but it is almost entirely absent in quiescent adult vasculature. However, in vessels undergoing angiogenesis, WT1 is dramatically upregulated. Studies have shown Wt1 has a role in both tumor and ischemic angiogenesis, but the mechanism of Wt1 action in angiogenic tissue remains to be elucidated. Here, we describe two methods for induction of in vivo angiogenesis (subcutaneous sponge implantation, femoral artery ligation) that can be used to assess the influence of Wt1 on new blood vessel formation. Subcutaneously implanted sponges s...
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

Isolation and Fluorescence-Activated Cell Sorting of Murine WT1-Expressing Adipocyte Precursor Cells
The current global obesity epidemic has triggered increased interest in adipose tissue biology. A major area of attention for many is adipose tissue development. A greater understanding of adipocyte ontogeny could be highly beneficial in answering questions about obesity-associated disease. Recent work has shown that a proportion of mature adipocytes in visceral white adipose tissue are derived from Wt1-expressing adipocyte precursor cells. These adipocyte precursor cells reside within the adipose tissue itself, and are a constituent of the stromal vascular fraction (SVF), along with other, non-adipogenic, cell types. Cruc...
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

Isolation and Colony Formation of Murine Bone and Bone Marrow Cells
Adult homeostasis is dependent on normal Wt1 expression. Loss of Wt1 expression in adult mice causes rapid loss of the mesenchymal tissues, fat and bone, amongst other phenotypes. Bone and bone marrow mesenchymal stromal cells can be studied by cell isolation and expansion. The stemness of these cells can then be characterized by carrying out a colony-forming unit-fibroblast assay and observing clonogenic capabilities. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

Biological Systems and Methods for Studying WT1 in the Epicardium
The embryonic epicardium is an important source of cardiovascular precursor cells and paracrine factors required for adequate heart formation. During embryonic heart formation, WT1 is mainly expressed in epicardial cells and epicardial derived cells. Its expression has been used to trace epicardial derivatives in embryos and recently it has been used to follow the reactivation of epicardial cells after myocardial infarction. Interestingly, the highest level of expression of WT1 during epicardium development correlates with the highest proliferative state, stem cell properties, and migratory capacity of epicardial cells. He...
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

Tools and Techniques for Wt1-Based Lineage Tracing
We describe the presently used constitutive or inducible genetic lineage tracing approaches based on the Cre/loxP system utilizing Cre recombinase expression under control of a Wt1 promoter. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

The Role of WT1 in Embryonic Development and Normal Organ Homeostasis
The Wilms’ tumor suppressor gene 1 (Wt1) is critically involved in a number of developmental processes in vertebrates, including cell differentiation, control of the epithelial/mesenchymal phenotype, proliferation, and apoptosis. Wt1 proteins act as transcriptional and post-transcriptional regulators, in mRNA splicing and in protein–protein interactions. Furthermore, Wt1 is involved in adult tissue homeostasis, kidney function, and cancer. For these reasons, Wt1 function has been extensively studied in a number of animal models to establish its spatiotemporal expression pattern and the developmental fate of the...
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

Clinical Aspects of WT1 and the Kidney
For more than 30 years, WT1 mutations have been associated with complex developmental syndromes involving the kidney. Acting as a transcription factor, WT1 is expressed throughout the nephron and controls the reciprocal interactions and phenotypic changes required for normal renal development. In the adult, WT1 expression remains extremely high in the renal podocyte, and at a lower level in the parietal epithelial cells. Wt1-null mice are unable to form kidneys [1]. Unsurprisingly, WT1 mutations lead to significant abnormalities of the renal and genitourinary tract, causing a number of human diseases including syndromes su...
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

WT1 Mutation in Childhood Cancer
In this chapter, the role of WT1 in childhood cancer is discussed, using the key examples Wilms’ tumor, desmoplastic small round cell of childhood, and leukemia. The role of WT1 in each disease is described and mirrored to the role of WT1 in normal development. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

Immunotherapy Targeting WT1: Designing a Protocol for WT1 Peptide-Based Cancer Vaccine
There is much current excitement about the potential of cancer immunotherapy. WT1 is high on the National Cancer Institute’s list of priority antigens for immune therapy. In this chapter we describe a protocol for a clinical trial using a WT1 peptide-based cancer vaccine. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

Bioinformatic Analysis of Next-Generation Sequencing Data to Identify WT1-Associated Differential Gene and Isoform Expression
Differential gene expression analysis has been conventionally performed by microarray techniques; however with the recent advent of next-generation sequencing (NGS) approaches, it has become easier to analyze the coding as well as the noncoding components. Additionally, NGS data analysis also provides information regarding the expression changes of specific isoforms. There are several bioinformatics tools available to analyze NGS data but with different parameters. This chapter provides a comparative insight into these tools by utilizing NGS datasets available from Wt1 knockout and embryonic stem cell line model. (Source: ...
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

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

Application of RNA-Seq Technology in Cancer Chemoprevention
RNA-sequencing is a revolutionary tool to follow differential expression after treatment with cancer chemopreventive agents. It allows a real genome-wide screening independent of prior assumptions and is well suited for analyzing coding but also long noncoding RNAs. It still consents the discovery of new genes and isoforms and increased our knowledge of antisense and other noncoding RNAs in a tremendous manner. Moreover, it permits to detect low-abundance and biologically critical isoforms and reveals genetic variants and gene fusions in one single assay. Here, we provide a detailed protocol for stranded RNA-sequencing. (S...
Source: Springer protocols feed by Cancer Research - November 28, 2015 Category: Cancer & Oncology Source Type: news

microRNAs in Cancer Chemoprevention: Method to Isolate Them from Fresh Tissues
microRNAs are 22-nucleotide-long double-strand small RNAs, able to modulate gene expression at posttranscriptional level, degrading mRNA and/or impairing translation. They have been shown to regulate mRNA and protein abundance and to participate in many regulatory circuits controlling developmental timing, cell proliferation and differentiation, apoptosis and stress response. Notably, microRNA activity has been correlated to the pathogenesis of cancer; they are aberrantly expressed in solid and hematological tumors, suggesting that they could function as oncogenes or tumor suppressors. The emerging role of miRNAs in the ca...
Source: Springer protocols feed by Cancer Research - November 28, 2015 Category: Cancer & Oncology Source Type: news

Use of Buffy Coat miRNA Profiling for Breast Cancer Prediction in Healthy Women
MicroRNAs are key regulators of different biological processes and their deregulation is associated with the occurrence of many diseases among which cancer. Due to the higher stability of microRNAs and to the easiness in their detection both in organs than in biological fluids, many studies are turned toward potential use of this small molecules as biomarkers for the prediction and diagnosis of different types of cancer. Here we describe the experiment protocol that we have used for microRNA profiling analysis in buffy coat samples of women who developed breast cancer versus women who remained healthy during a 20 year foll...
Source: Springer protocols feed by Cancer Research - November 28, 2015 Category: Cancer & Oncology Source Type: news

Controlled Delivery of Chemopreventive Agents by Polymeric Implants
The clinical development of cancer chemopreventive agents has been hampered by poor oral bioavailability issue. Several compounds have low aqueous solubility and undergo extensive first pass metabolism following oral dosing. To overcome this limitation, we developed polymeric implants from biodegradable ε-polycaprolactone (PCL) that can deliver both lipophilic as well as hydrophilic compounds. Implants furnish controlled release of compounds for long duration and provide dose-dependent release. The rate of release in vitro correlated well with the in vivo release. The polymeric implant technology thus overcomes the...
Source: Springer protocols feed by Cancer Research - November 28, 2015 Category: Cancer & Oncology Source Type: news

South African Herbal Extracts as Potential Chemopreventive Agents: Screening for Anticancer Splicing Activity
RT-PCR is an invaluable tool for the detection and characterization of mRNA. Cancer cell lines are treated with crude plant extracts and RNA is extracted and purified with DNase prior to RT-PCR. RT-PCR first-strand cDNA synthesis is done using random primers and can be refrigerated at 4 °C. PCR from the stored cDNA is performed using transcript-specific primers and electrophoresed on a molecular grade agarose gel to separate the splice variants. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - November 28, 2015 Category: Cancer & Oncology Source Type: news

Antibody Array as a Tool for Screening of Natural Agents in Cancer Chemoprevention
The efficacy of a given drug resides mainly on its ability to specifically target disease mechanisms. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - November 28, 2015 Category: Cancer & Oncology Source Type: news

3D Tumor Models and Time-Lapse Analysis by Multidimensional Microscopy
The 3D culture is advantageous in reflecting the in vivo condition compared to the 2D culture; however, imaging 3D-cultured cells may be a challenge due to technical restrictions. Recent development of confocal spinning disc microscope system as well as sophisticated software has enabled us to monitor dynamism of cell movement in multiple dimensions. Here we describe the method for time-lapse imaging of 3D-cultured cancer cells co-cultured with non-cancerous cells and discuss current limitations and future perspectives. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - November 28, 2015 Category: Cancer & Oncology Source Type: news

Protocol for a Steady-State FRET Assay in Cancer Chemoprevention
Cancer chemoprevention is an important strategy to prevent, reverse, or suppress the development of cancer. One of the target pathways that has emerged in recent years is the Keap1-Nrf2-ARE system that regulates the protection of cells against various carcinogens and their metabolites. Increased concentrations of the redox transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) induces the activation of antioxidant and phase 2 detoxifying genes. Nrf2 is regulated by substrate adaptor protein Kelch-like ECH-associated protein 1 (Keap1) that can target Nrf2 for ubiquitination and degradation by the proteasome...
Source: Springer protocols feed by Cancer Research - November 28, 2015 Category: Cancer & Oncology Source Type: news

Autophagy in Cancer Chemoprevention: Identification of Novel Autophagy Modulators with Anticancer Potential
Cancer cells have the ability to tolerate extreme conditions, autophagy-related stress tolerance enables cancer cells to survive by maintaining energy production that leads to cell growth and therapeutic resistance. Insufficient activation of autophagy in nutrient-deprived cancer cells may sensitize cancer cells to a broad array of chemotherapeutic agents and ionizing radiation. Therefore, identification of novel autophagy modulators with lower toxicity and better therapeutic index would be beneficial for cancer therapy. Here, we describe several currently used biochemical methods to assess autophagic activity and lysosoma...
Source: Springer protocols feed by Cancer Research - November 28, 2015 Category: Cancer & Oncology Source Type: news

Isolation of Chemoresistant Cell Subpopulations
Chemoresistance is a major challenge for cancer therapy and drives tumor relapse. The emergence, within the treated tumor mass, of specific cancer cell subpopulations endowed with high tolerance to the microenvironment stress induced by therapy is being growingly recognized as a mechanism of tumor progression. To obtain detailed information with regard to the pathways underlying survival, expansion, and microenvironmental cross talk of such chemoresistant cell subpopulations may be instrumental for cancer chemoprevention. Additionally, the obtained cell subpopulations may be used for direct screening of cancer chemoprevent...
Source: Springer protocols feed by Cancer Research - November 28, 2015 Category: Cancer & Oncology Source Type: news

Cancer Gastric Chemoprevention: Isolation of Gastric Tumor-Initiating Cells
Gastric cancer is an important healthcare problem and represents the second leading cause of death for malignant disease worldwide. In the Western world, the diagnosis is done at late stage when treatments can be only palliative. Searches for new therapeutic regimens as well as for new biomarkers are in progress. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - November 28, 2015 Category: Cancer & Oncology Source Type: news

AlgiMatrix™-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 - November 28, 2015 Category: Cancer & Oncology Source Type: news

Angiogenesis Assays
Neoangiogenesis constitutes one of the first steps of tumor progression beyond a critical size of tumor growth, which supplies a dormant mass of cancerous cells with the required nutrient supply and gaseous exchange through blood vessels essentially needed for their sustained and aggressive growth. In order to understand any biological process, it becomes imperative that we use models, which could mimic the actual biological system as closely as possible. Hence, finding the most appropriate model is always a vital part of any experimental design. Angiogenesis research has also been much affected due to lack of simple, reli...
Source: Springer protocols feed by Cancer Research - November 28, 2015 Category: Cancer & Oncology Source Type: news

Methods for Assessing the In Vivo Role of PTEN in Glucose Homeostasis
We describe the generation of tissue-specific PTEN knockout mice and models of both type 1 and type 2 diabetes, which we have found useful for the study of diabetes pathogenesis. We also outline common methods suitable for the characterization of glucose homeostasis in rodent models, including techniques to measure beta cell function and insulin sensitivity. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - November 18, 2015 Category: Cancer & Oncology Source Type: news

Germline PTEN Mutation Analysis for PTEN Hamartoma Tumor Syndrome
Clinically, deregulation of PTEN function resulting in reduced PTEN expression and/or activity is implicated in human disease. Cowden syndrome (CS) is an autosomal dominant disorder characterized by benign and malignant tumors. CS-related individual features occur commonly in the general population. Approximately 25 % of patients diagnosed with CS have pathogenic germline PTEN mutations, which increase lifetime risks of breast, thyroid, uterine, renal, and other cancers. PTEN testing and intensive cancer surveillance allow for early detection and treatment of these cancers for mutation-positive patients and their relatives...
Source: Springer protocols feed by Cancer Research - November 18, 2015 Category: Cancer & Oncology Source Type: news

Detecting PTEN and PI3K Signaling in Brain
The central nervous system is comprised of multiple cell types including neurons, glia, and other supporting cells that may differ dramatically in levels of signaling pathway activation. Immunohistochemistry in conjunction with drug interference are powerful tools that allow evaluation of signaling pathways in different cell types of the mouse central nervous system in vivo. Here we provide detailed protocols for immunohistochemistry to evaluate three essential components in the PI3K pathway in mouse brain: Pten, p-Akt, and p-4ebp1, and for rapamycin treatment to modulate mTOR signaling in vivo. (Source: Springer protocols...
Source: Springer protocols feed by Cancer Research - November 18, 2015 Category: Cancer & Oncology Source Type: news

Measurement of PTEN by Flow Cytometry
Recent advancements have driven the development of smaller footprint, less expensive, and user-friendly flow cytometers introducing the technology to more users. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - November 18, 2015 Category: Cancer & Oncology Source Type: news

Immunopathologic Assessment of PTEN Expression
Immunohistochemistry (IHC) is an excellent technique used routinely to define the phenotype in pathology laboratories through the analysis of molecular expression in cells and tissues. The PTEN protein is ubiquitously expressed in the majority of human tissues, and allelic or complete loss of PTEN is frequently observed in different types of malignancies leading to an activation of the AKT/mTOR pathways. IHC-based analyses are best to determine the level of PTEN expression in histological samples, but not to assess partial or heterozygous deletions, for which FISH analyses are more appropriate. Interpretation of the IHC re...
Source: Springer protocols feed by Cancer Research - November 18, 2015 Category: Cancer & Oncology Source Type: news

PTEN at 18: Still Growing
Discovered in 1997, PTEN remains one of the most studied tumor suppressors. In this issue of Methods in Molecular Biology, we assembled a series of papers describing various clinical and experimental approaches to studying PTEN function. Due to its broad expression, regulated subcellular localization, and intriguing phosphatase activity, methodologies aimed at PTEN study have often been developed in the context of mutations affecting various aspects of its regulation, found in patients burdened with PTEN loss-driven tumors. PTEN’s extensive posttranslational modifications and dynamic localization pose unique challeng...
Source: Springer protocols feed by Cancer Research - November 18, 2015 Category: Cancer & Oncology Source Type: news

PTEN: History of a Tumor Suppressor
Starting from the discovery of “inhibitory chromosomes” by Theodor Boveri to the finding by Henry Harris that fusing a normal cell to a cancer cell reduced tumorigenic potential, the notion of tumor suppression was recognized well before any tumor-suppressor genes were discovered. Although not the first to be revealed, PTEN has been demonstrated to be one of the most frequently altered tumor suppressors in cancer. This introductory chapter provides a historical perspective on our current understanding of PTEN including some of the seminal discoveries in the tumor suppressor field, the events leading to PTEN&rsq...
Source: Springer protocols feed by Cancer Research - November 18, 2015 Category: Cancer & Oncology Source Type: news