Techniques In Cell Cycle Analysis

Quantification of the proliferative characteristics of normal and malignant cells has been of interest to oncolo gists and cancer biologists for almost three decades. This interest stems from (a) the fact that cancer is a disease of uncontrolled proliferation, (b) the finding that many of the commonly used anticancer agents are preferentially toxic to cells that are actively proliferating, and (c) the observa tion that significant differences in proliferation characteristics exist between normal and malignant cells. Initially, cell cycle analysis was pursued enthusiastically in the hope of gener ating information useful for the development of rational cancer therapy strategies; for example, by allowing identi fication of rapidly proliferating tumors against which cell cycle-specific agents could be used with maximum effec tiveness and by allowing rational scheduling of cell cyc- specific therapeutic agents to maximize the therapeutic ratio. Unfortunately, several difficulties have prevented realiza tion of the early promise of cell cycle analysis: Proliferative patterns of the normal and malignant tissues have been found to be substantially more complex than originally an ticipated, and synchronization of human tumors has proved remarkably difficult. Human tumors of the same type have proved highly variable, and the cytokinetic tools available for cell cycle analysis have been labor intensive, as well as somewhat subjective and in many cases inapplicable to humans. However, the potential for substantially improved cancer therapy remains if more accurate cytokinetic infor mation about human malignancies and normal tissues can be obtained in a timely fashion.

This volume covers a broad range of cell types including cultured cell lines, primary cells, and various unicellular organisms such as fission yeast, budding yeast, parasite Leishmania amazonensis, and parasite Trypanosoma brucei. The chapters in this book are organized into four parts. Part One looks at a general overview of cell cycle control and synchronization. Part Two discusses techniques to synchronize mammalian cells to various cell cycle phases including mitotic sub-phases. Part Three covers synchronization of unicellular organisms and Part Four analyzes cell cycle progression. 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 thorough, Cell-Cycle Synchronization: Methods and Protocols is a valuable resource for both novice and expert scientists in this developing field.

Cell Cycle Control and Dysregulation Protocols focuses on emerging methodologies for studying the cell cycle, kinases, and kinase inhibitors. It addresses the issue of gene expression in vivo and in vitro, the analysis of cyclin-dependent kinase inhibitors, protein degradation mediated by the proteosome, the analysis of the transformed cell phenotype, and innovative techniques to detect apoptosis. Because there are already many manuals and protocols available, along with commercial kits and reagents, a variety of the more common techniques have not been included in our book. The protocols described, based on rather sophisticated techniques for in vivo and in vitro studies, consist of molecular biology, biochemistry, and various types of immunoassays. Indeed, the authors have successfully accomplished an arduous task by presenting several topics in the simplest possible manner. We are confident that Cell Cycle Control and Dysregulation Protocols will facilitate and optimize the work of practical scientists involved in researching the cell cycle. We greatly acknowledge the extraordinary contribution of the authors in writing this book.

The fundamental question of how cells grow and divide has perplexed biologists since the development of the cell theory in the mid-19th century, when it was recognized by Virchow and others that “all cells come from cells.” In recent years, considerable effort has been applied to the identification of the basic molecules and mechanisms that regulate the cell cycle in a number of different organisms. Such studies have led to the elucidation of the central paradigms that underpin eukaryotic cell cycle control, for which Lee Hartwell, Tim Hunt, and Paul Nurse were jointly awarded the Nobel Prize for Medicine and Physiology in 2001 in recognition of their seminal contributions to this field. The importance of understanding the fundamental mechanisms that modulate cell division has been reiterated by relatively recent discoveries of links between cell cycle control and DNA repair, growth, cellular metabolism, development, and cell death. This new phase of integrated cell cycle research provides further challenges and opportunities to the biological and medical worlds in applying these basic concepts to understanding the etiology of cancer and other proliferative diseases.

The field of cell cycle regulation is based on the observation that the life cycle of a cell progresses through several distinct phases, G1, M, S, and G2, occurring in a well-defined temporal order. Details of the mechanisms involved are rapidly emerging and appear extraordinarily complex. Furthermore, not only is the order of the phases important, but in normal eukaryotic cells one phase will not begin unless the prior phase is completed successfully. Che- point control mechanisms are essentially surveillance systems that monitor the events in each phase, and assure that the cell does not progress prematurely to the next phase. If conditions are such that the cell is not ready to progress—for example, because of incomplete DNA replication in S or DNA damage that may interfere with chromosome segregation in M—a transient delay in cell cycle progression will occur. Once the inducing event is properly handled— for example, DNA replication is no longer blocked or damaged DNA is repaired—cell cycle progression continues. Checkpoint controls have recently been the focus of intense study by investigators interested in mechanisms that regulate the cell cycle. Furthermore, the relationship between checkpoint c- trol and carcinogenesis has additionally enhanced interest in these cell cycle regulatory pathways. It is clear that cancer cells often lack these checkpoints and exhibit genomic instability as a result. Moreover, several tumor suppressor genes participate in checkpoint control, and alterations in these genes are as- ciated with genomic instability as well as the development of cancer.

​ This volume brings together a unique collection of protocols that cover standard, novel, and specialized techniques. Cell Cycle Oscillators: Methods and Protocols guides readers through recent progress in the field from both holistic and reductionist perspectives, providing the latest developments in molecular biology techniques, biochemistry, and computational analysis used for studying oscillatory networks. 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. Authoritative and cutting-edge, Cell Cycle Oscillators: Methods and Protocols will serve as an invaluable reference to gain further insight into the complex and incompletely understood processes that are involved in the cell cycle and its regulation by oscillatory networks.