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Since the discovery of p53 as a tumor suppressor, numerous methods have evolved to reveal the unique structural features and biochemical functions of this protein. Several unique properties of p53 posed a challenge to understa- ing its normal function in the initial phase of its research. The low levels of p53 in normal cells, its stabilization under situations of genotoxic stress, induction of growth arrest, and apoptosis with stabilization of the protein, obstructed the visibility of its normal, unmutated function. The property of p53 that can sense a promoter and transactivate or inhibit is still not well understood. It is still not known whether it is the absence of the protein that causes tumorigenesis, or if its mutants have a dominant role in inducing cancer. p53 Protocols comprises eighteen chapters for the study of the diverse properties of p53 and related proteins. The methods included are invaluable for delineating the function of other proteins that may function as tumor suppr- sors or growth suppressors. The chapters are not presented in any schematic order, for the importance and diversity of the functions of p53 make it imp- sible to organize them suitably. We have made a sincere effort to collect the methods most useful to those investigators working on tumor suppressors or growth suppressors. The purpose of p53 Protocols is not only to provide investigators with methods to analyze similar biochemical functions, but also to familiarize them with the associated problems that arose during the course of investigations.
Protocols used in Molecular Biology is a compilation of several examples of molecular biology protocols. Each example is presented with a concise introduction, materials and chemicals required, a step-by-step procedure and troubleshooting tips. Information about the application of the protocol is also provided. The techniques included in this book are essential to research in the fields of proteomics, genomics, cell culture, epigenetic modification and structural biology. The protocols can also be used by clinical researchers (neuroscientists and oncologists, for example) for medical applications (diagnostics, therapeutics and multidisciplinary projects).
Decades of research on the tumor suppressor p53 have revealed that it plays a significant role as a "guardian of the genome," protecting cells against genotoxic stress. In recent years, p53 research has begun to move into the clinic in attempts to understand how p53 is frequently inactivated in-and sometimes even promotes-human cancer. Written and edited by experts in the field, this collection from Cold Spring Harbor Perspectives in Medicine covers the rapid progress that has recently been made in basic and clinical research on p53. The contributors review new observations about its basic biology, providing updates on the functions of its isoforms and domains, the myriad stresses and signals that trigger its activation or repression, and its downstream effects on genome stability and the cell cycle that enforce tumor suppression in different cell and tissue types. They also discuss how p53 dysfunction contributes to cancer, exploring the various inherited and somatic mutations in the human TP53 gene, the impact of mutant p53 proteins on tumorigenesis, and the prognostic value and clinical outcomes of these mutations. Drugs that are being developed to respond to tumors harboring aberrant p53 are also described. This book is therefore essential reading for all cancer biologists, cell and molecular biologists, and pharmacologists concerned with the treatment of this disease.
A collection of cutting-edge techniques for analyzing genotoxic exposure and detecting the resulting biological effects-including endogenous metabolites-up to and including the development of cancer. The authors emphasize analytical methods that can be specifically applied to human populations and patients. Among the applications detailed are the analysis of interactions between such cellular macromolecules as DNA and proteins and chemical and physical agents, the assessment of medically relevant toxicity, and the characterization of genetic alterations induced in transgenic animals by in vivo systems. There are also methods for the analysis of genotoxic exposure during gene expression, of cytotoxicity caused by the induction of apoptosis, of genetic alterations in reporter genes and oncogenes, early (premalignant) detection of altered oncogenes, and of individual variation in biotransformation and DNA repair capacity.
This volume presents the latest collection of immunophenotypic techniques and applications used in research and clinical settings. Chapters in this book cover topics such as constructions of high dimensions fluorescence and mass cytometry panels; fluorescence barcoding; using dried or lyophilized reagents; and immunophenotypic examples of specific cell types. The book concludes with a discussion on the critical roles of quality control and immunophenotyping in the clinical environment. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and comprehensive, Immunophenotyping: Methods and Protocols is a valuable resource for any researchers, clinician, or scientist interested in learning more about this evolving field.
This volume offers a comprehensive review of the functions of the p53 family. The contributors examine the normal roles of these transcription factors, their evolution, the regulatory mechanisms that control p53 activity, and the part played by p53 mutations in tumorigenesis.
The various cell types have traditionally been recognized and classified according to their appearance in the light microscope following the process of fixing, processing, sectioning, and staining tissues that is known as histology. Classical histology has been augmented by immunohistochemistry (the use of specific antibodies to stain particular molecular species in situ). Immunohistochemistry has allowed the identification of many more cell types than could be visualized by classical histology, particularly in the immune system and among the scattered hormone-secreting cells of the endocrine system. Handbook of Immunohistochemistry and in Situ Hybridization of Human Carcinomas discusses all aspects of immunohistochemistry and in situ hybridization technologies and the important role they play in reaching a cancer diagnosis. It provides step-by-step instructions on the methods of additional molecular technologies such as DNA microarrays, and microdissection, along with the benefits and limitations of each method. The topics of region-specific gene expression, its role in cancer development and the techniques that assist in the understanding of the molecular basis of disease are relevant and necessary in science today, ensuring a wide audience for this book. - The only book available that translates molecular genetics into cancer diagnosis - Provides the readers with tools necessary to perform and optimize sensitive, powerful techniques, including immunohistochemistry and fluorescence in situ hybridization, used in tumor diagnosis - Written by experts in this field, the book provides theoretical considerations as well as practical approaches to carry out effectively these techniques - Offers suggestions, tips, cautions, and guidelines to avoid artifacts and misdiagnosis - Introduces new techniques to detect genes and proteins involved in the initiation and progression of cancer - Covers the latest developments and a wide range of applications to the detection of antigens and single-copy DNA and RNA - Written in a uniform format, each chapter includes Introduction, Materials required, step-by-step detailed Methods, Results, Discussion, and comprehensive up-to-date References
It has become clear that tumors arise from excessive cell proliferation and a c- responding reduction in cell death. Tumors result from the successive accumulation of mutations in key regulatory target genes over time. During the 1980s, a number of oncogenes were characterized, whereas from the 1990s to the present, the emphasis shifted to tumor suppressor genes (TSGs). It has become clear that oncogenes and tumor suppressor genes function in the same pathways, providing positive and ne- tive growth regulatory activities. The signaling pathways controlled by these genes involve virtually every process in cell biology, including nuclear events, cell cycle, cell death, cytoskeletal, cell membrane, angiogenesis, and cell adhesion effects. Tumor suppressor genes are mutated in hereditary cancer syndromes, as well as somatically in nonhereditary cancers. In their normal state, TSGs control cancer development and p- gression, as well as contribute to the sensitivity of cancers to a variety of therapeutics. Understanding the classes of TSGs, the biochemical pathways they function in, and how they are regulated provides an essential lesson in cancer biology. We cannot hope to advance our current knowledge and to develop new and more effective therapies without understanding the relevant pathways and how they influence the present approaches to therapy. Moreover, it is important to be able to access the powerful tools now available to discover these genes, as well as their links to cell biology and growth control.
Since the first edition of this book dedicated to differential display (DD) technology was published in 1997, we have witnessed an explosive interest in studying differential gene expression. The gene-hunting euphoria was initially powered by the invention of DD, which was gradually overtaken by DNA microarray technology in recent years. Then why is there still the need for second edition of this DD book? First of all, DD still enjoys a substantial lead over DNA microarrays in the ISI citation data (see Table 1), despite the h- dreds of millions of dollars spent each year on arrays. This may come as a surprise to many, but to us it implies that many of the DNA microarray studies went unpublished owing to their unfulfilled promises (1). Second, unlike DNA microarrays, DD is an “open”-ended gene discovery method that does not depend on prior genome sequence information of the organism being studied. As such, DD is applicable to the study of all living organisms—from bacteria, fungi, insects, fish, plants, to mammals—even when their genomes are not sequenced. Second, DD is more accessible technically and financially to most cost-conscious “cottage-industry” academic laboratories. So clearly DD still has its unique place in the modern molecular biological toolbox for gene expression analysis.