Transgenic Mouse Models

Transgenic mice technology has provided a powerful tool for both neoplastic and non-neoplastic disease investigation. Injection of exogenous DNA into fertilized eggs (zygotes) is known as trangenesis and is the most common method to modify the germline of mice. The offspring of transgenic founders are usually examined for a specific phenotype and are used to characterize genes and gene regulatory elements. The procedure involves a construct made of a segment of DNA that contains the sequence of...

Proteomics Instrumentation

The first protein separation method that met omics criteria was two-dimensional polyacrylamide gel electrophoresis (2D PAGE). This technique is able to accept minimally prepared specimens and to separate all proteins present in the mixture. Because of its contribution to proteomics and its popularity among researchers, the name two-dimensional (2D) electrophoresis has become almost synonymous with the term proteomics. Two-dimensional PAGE includes two procedural steps. The first is isoelectric...

Larry Fowler and Wieslaw Furmaga

Omics is a term designating a complete analysis of biological systems in which entire metabolic pathways are studied. Omics changes the methodological approach from small-scale research of one gene, one protein, or one metabolic reaction to the large comprehensive level of synthetic study in which the entire genome, protein composition, or metabolic pathway is studied simultaneously. The main breakthrough to the omics era was a confirmation of the complete human genome sequence. Entering this...

Epimutations in Cancer

Neoplastic transformation is associated with alterations in DNA methylation, including both global hypomethylation and gene-specific hypermethylation.197-199 Gains of DNA methylation in cancer cells typically reflect hypermethyla-tion of CpG islands in gene promoter regions, which contributes to gene silencing. 1 97 Methylation-dependent gene silencing is a normal mechanism for regulation of gene expression.200 However, in cancer cells methylation-dependent epigenetic gene silencing represents...

LOH in Benign Diseases

There is evidence to suggest that benign conditions, such as chronic obstructive pulmonary disease (COPD) and asthma, have a genetic predisposition and probably arise via the interaction between multiple gene products.126 Studies of microsatellite instability and LOH have been employed to study these diseases with the goals of identifying causative genes and or develop genetic screening tools for use in epidemiology. LOH in several prevalent noncancer diseases is discussed next. LOH in...

Conclusions

Molecular tests that directly analyze nuclei acids can provide highly specific diagnostic and predictive information, but IHC continues to play an essential role in biomarker testing and clinical decision making. Because the quantitative aspect of predictive marker testing by IHC is subject to significant analytical variability, steps must be taken to ensure that standardized methods are used throughout the testing process. 1. Early Breast Cancer Trialists' Collaborative Group (EBCTCG). Effects...

Array CGH Platforms

Arrays Based on Clone Inserts cDNA Arrays The first genome-wide application of array CGH was based on cDNA arrays, with each spot representing one reversely transcribed mRNA.5 Apart from the fact that these arrays were readily available, the main advantage of cDNA arrays was that they facilitated a direct comparison of DNA copy number changes with gene expression data derived from the same tumor.i7 However, cDNA arrays are extremely gene focused and thus only a small percentage of the genome is...

Advantages and Limitations of Tissue Culture

Tissue culture has become the penultimate tool of the reductionist biologist. The utilization of tissue culture as a research methodology has allowed investigators to study isolated interactions in its near-normal environment. These experiments by their very nature introduce artifacts however, they do minimize the number of confounding variables that may affect a particular experiment. For instance, tissue culture allows investigators to determine the effects of one particular treatment on a...

Role of Immunohistochemistry in Biomarker Testing

For some markers, such as steroid hormone receptors in breast cancer, IHC is the standard method of identifying patients likely to respond to specific drugs. Patients with tumors that are estrogen receptor positive ER(+) (about 70 of breast cancers) are more likely to respond to selective estrogen receptor modulators (e.g., tamox-ifen) or aromatase inhibitors than those with ER(-) tumors. Indeed, ER expression as assessed by IHC has been shown to be the single most important biomarker in breast...

Chromogenic In Situ Hybridization CISH

CISH is another type of in situ hybridization that is very similar to FISH except that the probe is visualized by a chromogenic reaction after it has hybridized to its target rather than being visualized as a fluorescent signal. There are several ways in which one can perform CISH. One method incorporates biotinylated-dUTP into the probe. After hybridization of the probe to its target, horseradish peroxidase-labeled streptavidin is added to the slide. The streptavidin attaches to the biotin....

Extrinsic Death Pathway

The extrinsic pathway involves binding of death ligands such as tumor necrosis factor-a (TNF-a), CD95 ligand (Fas ligand), and TNF-related apoptosis-inducing ligand (TRAIL) to their cognate cell-surface receptors TNFR1, CD95'Fas, TRAIL-R1, TRAIL-R2, and the DR series of receptors,5 0 resulting in the activation of initiator caspase-8 (also known as FADD-homologous ICE'CED-3-like protease, or FLICE) and subsequent activation of effector caspase-3 (see Fig. 4.2 ).51 The cytoplasmic domains of...

Contributors

Department of Pathology, University of Texas Health Science Center at Tyler, Tyler, TX, USA Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, New York, NY, USA The Methodist Hospital, Houston, TX, USA Gregory L. Blakey Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Pathology and Laboratory Medicine, Weill Medical College of Cornell University, New York, NY, USA The Methodist Hospital, Houston, TX, USA...

Three Dimensional Models of Lung Disease

Current cell culture models have shortcomings resulting in unreliable tumor growth, uncharacteristic tumor development, nonhuman tumors, and inadequate methods of detection. Cells propagated under traditional culture conditions differ widely in their expression of differentiated markers, adhesion receptors, and growth factor receptors compared with cells in situ or those grown as tissue-like structures.90,91 This factor is of concern because the phenotypic changes leading to malignant...

Data Analysis and Interpretation

At the time of flow cytometry analysis, it is important to collect data on sufficient events cell numbers and all available parameters to ensure detection of the cells of interest. The immunophenotyping characterization of hematopoietic and lymphoid neoplasms relies solidly on the principle that neoplastic cells frequently exhibit abnormal or aberrant antigenic expression that differs from normal cells.20-22 Abnormal aberrant antigenic expression can be grouped into four basic categories...

Gene Mutations in Cancer

Mutations affecting a variety of genes have been characterized in cancer. Some of these mutations represent activating mutations of proto-oncogenes or other positive mediators of cell proliferation and others represent inactivating mutations of tumor suppressor genes or other negative mediators of cell proliferation . Mutations in these genes synergize with other genetic chromosomal and epigenetic abnormalities to drive neoplastic transformation and tumorigenesis in affected cells and tissues....

Selectins

The selectin family of adhesion molecules, consisting of L eukocyte selectin, E ndothelial selectin, and P latelet selectin, are calcium-dependent type I transmembrane glyco-proteins with extracellular lectin-like domains that interact, for example, with sialylated carbohydrate-determinant and mucin-like glycoproteins.121 They have been studied predominantly by examination of the recruitment of leukocytes from the circulation.4,121 Adhesion with selectins is calcium dependent, and the ligands...

In Situ Hybridization General Principles

Phosphate Sugar Thymine

To understand how in situ hybridization works, one should be familiar with 1 DNA and RNA composition and structure, 2 principles of base pairing, 3 denaturation and hybridization, and 4 factors that influence denaturation and hybridization. It is not possible to cover these topics extensively however, the following paragraphs briefly touch on the most important aspects of these topics. DNA and RNA are each composed of four nucleotides that form long chains. The nucleotides that comprise DNA...

Human Type II Alveolar Pneumocytes and Acute Lung Injury Acute Respiratory Distress Syndrome

Pulmonary alveolar type II cells are a unique cell subset that carries out highly specialized functions which include synthesis and secretion of surfactant, a unique composition of lipoproteins that act to reduce surface tension at the alveolar air-liquid interface.28 Defining the molecular mechanisms leading to production of surfactant by type II pneumocytes is important in many disease processes. The pathogenic sequence that results in ARDS, the most severe manifestation of alveolar lung...

CXCR4 Chemokine Receptor

Cancer cells must pass through blood or lymphatic circulation and through their vessel walls to metastasize. Metastatic tumor cells are thought to co-opt signals normally controlling leukocyte movement, such as chemokine-mediated cell migration.94 Emigration from vessels is regulated by a sequence of distinct molecular signals, one of which involves chemokines that activate integrins and direct migration of leukocytes.34 The chemokine stromal cell-derived factor-1 SDF-i CXCLi2 is a CXC...

Three Dimensional Biology

Human Small Intestine Scanning

Conventional Bioreactors and Three-Dimensionality The Origins of Three-Dimensional Culture Carrel postulated that tissue development was linked to access to nutrient supply, noting that peripheral cells grew readily, and internal cells became necrotic, presumably based on their distance from the nutrient source. To circumvent this issue, Carrel implemented cultures on silk veils, preventing the plasma clots of the growth media from deforming or becoming spherical, thus facilitating the internal...

Polymerase Chain Reaction

Pcr Slippage Polymerase

Polymerase chain reaction PCR enables one to determine if a specific needle is present in a haystack, and it can be used as a step toward the characterization of the needle. It is a quick, powerful, inexpensive DNA amplification technique that has become a fundamental tool in molecular pathology. The PCR is one of the most significant technical innovations in molecular biology.1 The PCR was devised by Kary Mullis and colleagues2,3 at Cetus Corporation in California and was first described in a...

Reverse Transcription

Reverse Transcriptase

All normal cells in a human's body, with few exceptions, have the same chromosomal DNA sequence, that is, the same genetic code. Thus, genomic information obtained from the DNA of easily obtained normal white blood cells would be applicable to the genetic makeup of normal lung, brain, or colon cells. This idea does not apply to malignant cells, which can have a genetic composition that is profoundly different from that of normal cells. Function and structure of various cell types differ because...

Timothy Craig Allen MD JD

University of Texas Health Science Center at Tyler Tyler, Texas Weill Medical College of Cornell University Timothy Craig Allen, MD, JD Department of Pathology University of Texas Health Science Center at Tyler Tyler, TX Weill Medical College of Cornell University ISBN 978-0-387-89625-0 e-ISBN 978-0-387-89626-7 Springer Dordrecht Heidelberg London New York Library of Congress Control Number 2008939864 Springer Science Business Media, LLC 2009 All rights reserved. This work may not be translated...

Protein Complexes and Protein Interactions

1n a physiological environment, proteins do not exist as single entities but rather as a complex system responsible for cellular function and regulation. The methods of studying protein complexes must be able to evaluate not one but multiple elements, as one-on-one interaction between proteins is more an exception than a rule. Proteins interact not only with other proteins but also with nucleic acids, small molecules, and other components, such as drugs. The classic low-throughput system for...