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The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with more than 300 volumes (all of them still in print), the Series contains much material still relevant today - truly an essential publication for researchers in all fields of life sciences.

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Publications in this journal

• Studies of enzymatic cleavage of cellulose using Polysaccharide Analysis by Carbohydrate gel Electrophoresis (PACE)

  Authors: Ondrej Kosik, Jennifer Bromley, Marta Busse, Zhinong Zhang, Paul Dupree

  Methods in Enzymology.

• Selective fluorogenic derivatization of 3-nitrotyrosine and 3,4-dihydroxyphenylalanine in peptides: a method designed for quantitative proteomic analysis.

  Authors: Victor S Sharov, Elena S Dremina, Justin Pennington, Jacque Killmer, Christopher Asmus, Maria Thorson, Sung Jung Hong, Xiaobao Li, John F Stobaugh, Christian Schöneich

  Methods in enzymology. 441:19-32.

  There is a need for the selective derivatization and enrichment of posttranslational protein modifications from tissue samples. This chapter describes a method for the selective derivatization ofThere is a need for the selective derivatization and enrichment of posttranslational protein modifications from tissue samples. This chapter describes a method for the selective derivatization of 3-nitrotyrosine (after reduction to 3-amino-tyrosine) and 3,4-dihydroxyphenylalanine with benzylamine derivatives to yield 6-amino- and 6-benzylamine-substituted benzoxazoles, which display characteristic fluorescence properties. The methodology can be expanded to other substituted benzylamines, which carry functional groups for affinity enrichment.

• In-gel detection of S-nitrosated proteins using fluorescence methods.

  Authors: Nicholas J Kettenhofen, Xunde Wang, Mark T Gladwin, Neil Hogg

  Methods in enzymology. 441:53-71.

  Gel-based detection of protein S-nitrosothiols has relied on the biotin-switch method. This method attempts to replace the nitroso group with a biotin label to allow detection and isolation ofGel-based detection of protein S-nitrosothiols has relied on the biotin-switch method. This method attempts to replace the nitroso group with a biotin label to allow detection and isolation of S-nitrosated proteins and has been used extensively in the literature. This chapter describes a modification of this method that differs from the original in two major ways. First, it uses a combination of copper ions and ascorbate to achieve selective reduction of the S-nitrosothiol. Second, it replaces the biotin label with fluorescent cyanine dyes in order to directly observe the modified proteins in-gel and perform comparative studies using difference gel electrophoresis analysis in two dimensions.

• Practical approaches to investigate redox regulation of heat shock protein expression and intracellular glutathione redox state.

  Authors: Vittorio Calabrese, Anna Signorile, Carolin Cornelius, Cesare Mancuso, Giovanni Scapagnini, Bernardo Ventimiglia, Nicolo' Ragusa, Albena Dinkova-Kostova

  Methods in enzymology. 441:83-110.

  The products of vitagenes such as heat shock protein 32 (Hsp32, heme oxygenase 1) and Hsp70, the family of inducible cytoprotective proteins regulated by the Keap1/Nrf2/ARE pathway, and smallThe products of vitagenes such as heat shock protein 32 (Hsp32, heme oxygenase 1) and Hsp70, the family of inducible cytoprotective proteins regulated by the Keap1/Nrf2/ARE pathway, and small molecule antioxidants such as glutathione provide the cell with powerful means to counteract and survive various conditions of stress. Among these protective systems, the heat shock proteins represent a highly conserved and robust way for preservation of correct protein conformation, recovery of damaged proteins, and cell survival. Their regulation is dependent on the redox status of the cell, thus redox regulation is rapidly evolving as an important metabolic modulator of cellular functions, and is being increasingly implicated in many chronic inflammatory and degenerative diseases. Protein thiols play a key role in redox sensing, and regulation of cellular redox state is crucial mediator of multiple metabolic, signalling and transcriptional processes in the brain. Nitric oxide, and reactive nitrogen species induce the transcription of vitagenes and Keap1/Nrf2/ARE-dependent genes whose functional products protect against a wide array of subsequent challenges. Emerging interest is now focusing on exogenous small molecules that are capable of activating these systems as a novel target to minimize deleterious consequences associated with free radical-induced cell damage, such as during neurodegeneration. This chapter describes methods that can be used to assess the expression of heat shock proteins and the cellular glutathione redox status and discusses their relevance to mechanisms modulating the onset and progression of neurodegenerative diseases.

• Determination of S-nitrosothiols in biological and clinical samples using electron paramagnetic resonance spectrometry with spin trapping.

  Authors: Paul G Winyard, Iona A Knight, Frances L Shaw, Sophie A Rocks, Claire A Davies, Paul Eggleton, Richard Haigh, Matthew Whiteman, Nigel Benjamin

  Methods in enzymology. 441:151-60.

  S-Nitroso moieties, such as the S-nitroso group within S-nitrosated albumin, constitute a potential endogenous reservoir of nitric oxide (NO.) in human tissues and other biological systems. Moreover,S-Nitroso moieties, such as the S-nitroso group within S-nitrosated albumin, constitute a potential endogenous reservoir of nitric oxide (NO.) in human tissues and other biological systems. Moreover, S-nitroso compounds are under investigation as therapeutic agents in humans. Therefore, it is important to be able to detect S-nitrosothiols (RSNOs) in human extracellular fluids, such as plasma and synovial fluid, as well as other biological samples. This chapter describes a method for the determination of S-nitrosothiols in biofluids. The method is based on electron paramagnetic resonance (EPR) spectrometry, in combination with spin trapping using a ferrous ion complex of the iron chelator N-methyl-d-glucamine dithiocarbamate under alkaline conditions. This iron complex mediates the decomposition of RSNO to NO., as well as spin trapping the generated NO.. The resulting spin adduct has a unique EPR signal that can be quantified.

• Kinetic studies on peroxynitrite reduction by peroxiredoxins.

  Authors: Madia Trujillo, Gerardo Ferrer-Sueta, Rafael Radi

  Methods in enzymology. 441:173-96.

  Peroxiredoxins catalytically reduce peroxynitrite to nitrite. The peroxidatic cysteine of peroxiredoxins reacts rapidly with peroxynitrite. The rate constant of that reaction can be measured using aPeroxiredoxins catalytically reduce peroxynitrite to nitrite. The peroxidatic cysteine of peroxiredoxins reacts rapidly with peroxynitrite. The rate constant of that reaction can be measured using a stopped flow spectrophotometer either directly by following peroxynitrite disappearance in the region of 300 to 310 nm using an initial rate approach or steady-state measurements or by competition with a reaction of known rate constant. The reactions used to compete with peroxiredoxins include the oxidation of Mn(III)porphyrins and horseradish peroxidase by peroxynitrite. Additionally, a method is described in which a hydroperoxide competes with peroxynitrite for the oxidation of peroxiredoxin. Moreover, a fluorescent technique for determining the kinetics of thioredoxin-mediated peroxiredoxin reduction, closing the catalytic cycle, is also described. All methods reviewed provide reliable values of rate constants and a combination of them can be used to provide further reassurance; applicability and advantages of the different methodologies are discussed.

• Tyrosine nitration, dimerization, and hydroxylation by peroxynitrite in membranes as studied by the hydrophobic probe N-t-BOC-l-tyrosine tert-butyl ester.

  Authors: Silvina Bartesaghi, Gonzalo Peluffo, Hao Zhang, Joy Joseph, Balaraman Kalyanaraman, Rafael Radi

  Methods in enzymology. 441:217-36.

  Protein tyrosine oxidation mechanisms in hydrophobic biocompartments (i.e., biomembranes, lipoproteins) leading to nitrated, dimerized, and hydroxylated products are just starting to be appreciated.Protein tyrosine oxidation mechanisms in hydrophobic biocompartments (i.e., biomembranes, lipoproteins) leading to nitrated, dimerized, and hydroxylated products are just starting to be appreciated. This chapter reports on the use of the hydrophobic tyrosine analog N-t-BOC-l-tyrosine tert-butyl ester (BTBE) incorporated to phosphatidyl choline liposomes to study peroxynitrite-dependent tyrosine oxidation processes in model biomembranes. The probe proved to be valuable in defining the role of biologically relevant variables in the oxidation process, including the action of hydrophilic and hydrophobic peroxynitrite and peroxynitrite-derived free radical scavengers, transition metal catalysts, carbon dioxide, molecular oxygen, pH, and fatty acid unsaturation degree. Moreover, detection of the BTBE phenoxyl radical and relative product distribution yields of 3-nitro-, 3,3'-di-, and 3-hydroxy-BTBE in the membrane fully accommodate with a free radical mechanism of tyrosine oxidation, with physical chemical and biochemical determinants that in several respects differ of those participating in aqueous environments. The methods presented herein can be extended to explore the reaction mechanisms of tyrosine oxidation by other biologically relevant oxidants and in other hydrophobic biocompartments.

• Methods to measure the reactivity of peroxynitrite-derived oxidants toward reduced fluoresceins and rhodamines.

  Authors: Peter Wardman

  Methods in enzymology. 441:261-82.

  The commonest probes for "reactive oxygen and nitrogen species" are reduced fluorescein and rhodamine dyes that fluoresce when oxidized. The reduced dyes are reactive toward peroxynitrite, althoughThe commonest probes for "reactive oxygen and nitrogen species" are reduced fluorescein and rhodamine dyes that fluoresce when oxidized. The reduced dyes are reactive toward peroxynitrite, although probably not directly but via free radical oxidants derived from it: hydroxyl, carbonate, and nitrogen dioxide free radicals. The reaction with peroxynitrite can be monitored by rapid mixing and stopped-flow spectrophotometry, but reliable measurement of reactivity of the peroxynitrite-derived radicals requires specialized techniques such as flash photolysis or pulse radiolysis to monitor the fast reactions in real time. A key feature of oxidation by radicals is that the reaction produces an intermediate fluorescein or rhodamine radical, which normally is oxidized further by oxygen to yield the fluorescent, stable product. Susceptibility of the yield of fluorescence to interference by antioxidants can be assessed from kinetic parameters, which reflect reactivity. This chapter outlines methods for estimation of key rate constants involving peroxynitrite-derived oxidants.

• Reductive gas-phase chemiluminescence and flow injection analysis for measurement of the nitric oxide pool in biological matrices.

  Authors: Ulrike Hendgen-Cotta, Marijke Grau, Tienush Rassaf, Putrika Gharini, Malte Kelm, Petra Kleinbongard

  Methods in enzymology. 441:295-315.

  There is growing evidence for nitric oxide (NO.) being involved in cell signaling and pathology. Much effort has been made to elucidate and characterize the different biochemical reaction pathways ofThere is growing evidence for nitric oxide (NO.) being involved in cell signaling and pathology. Much effort has been made to elucidate and characterize the different biochemical reaction pathways of NO.in vivo. However, a major obstacle in assessing the significance of nitrosated species and oxidized metabolites often remains: a reliable analytical technique for the detection of NO. in complex biological matrices. This chapter presents refined methodologies, such as chemiluminescence detection and flow injection analysis, compared with adequate sample processing procedures to reliably quantify and assess the circulating and resident NO(.) pool, consisting of nitrite, nitrate, nitroso, and nitrosylated species.

• Interactive relations between nitric oxide (NO) and carbon monoxide (CO): heme oxygenase-1/CO pathway is a key modulator in NO-mediated antiapoptosis and anti-inflammation.

  Authors: Hun-Taeg Chung, Byung-Min Choi, Young-Guen Kwon, Young-Myeong Kim

  Methods in enzymology. 441:329-38.

  Nitric oxide (NO) and carbon monoxide (CO) are synthesized from l-arginine and heme by the catalytic reaction of NO synthase (NOS) and heme oxygenase (HO). NO, a highly reactive free radical, playsNitric oxide (NO) and carbon monoxide (CO) are synthesized from l-arginine and heme by the catalytic reaction of NO synthase (NOS) and heme oxygenase (HO). NO, a highly reactive free radical, plays an important role in the regulation of vascular and immune function, antiapoptosis, and neurotransmission by producing cGMP, nitrosyl iron complexes, and S-nitrosothiols. CO, a more stable molecule, exerts similar biological activities to those of NO by cGMP production, p38 mitogen-activated protein kinase activation, and nuclear factor-kappaB activation. NO induces the suppression of apoptosis and inflammation in hepatocytes and macrophages by an elevation in HO-1 and CO production, and these effects were not observed in mice lacking HO-1 as well as in cells treated with a HO-1 inhibitor. These evidences indicate that the HO-1/CO pathway is a key player in NO-mediated cytoprotection and anti-inflammation. This chapter reviews new advances in the interactive relations between iNOS/NO and HO-1/CO pathways in the regulation of apoptosis and inflammation.

• In vivo real-time measurement of nitric oxide in anesthetized rat brain.

  Authors: Rui M Barbosa, Cátia F Lourenço, Ricardo M Santos, Francois Pomerleau, Peter Huettl, Greg A Gerhardt, João Laranjinha

  Methods in enzymology. 441:351-67.

  During the last two decades nitric oxide (.NO) gas has emerged as a novel and ubiquitous intercellular modulator of cell functions. In the brain, .NO is implicated in mechanisms of synapticDuring the last two decades nitric oxide (.NO) gas has emerged as a novel and ubiquitous intercellular modulator of cell functions. In the brain, .NO is implicated in mechanisms of synaptic plasticity but it is also involved in cell death pathways underlying several neurological diseases. Because of its hydrophobicity, small size, and rapid diffusion properties, the rate and pattern of .NO concentration changes are critical determinants for the understanding of its diverse actions in the brain. .NO measurement in vivo has been a challenging task due to its low concentration, short half-life, and high reactivity with other biological molecules, such as superoxide radical, thiols, and heme proteins. Electrochemical methods are versatile approaches for detecting and monitoring various neurotransmitters. When associated with microelectrodes inserted into the brain they provide high temporal and spatial resolution, allowing measurements of neurochemicals in physiological environments in a real-time fashion. To date, electrochemical detection of .NO is the only available technique that provides a high sensitivity, low detection limit, selectivity, and fast response to measure the concentration dynamics of .NO in vivo. We have used carbon fiber microelectrodes coated with two layers of Nafion and o-phenylenediamine to monitor the rate and pattern of .NO change in the rat brain in vivo. The analytical performance of microelectrodes was assessed in terms of sensitivity, detection limit, and selectivity ratios against major interferents: ascorbate, dopamine, noradrenaline, serotonin, and nitrite. For the in vivo recording experiments, we used a microelectrode/micropipette array inserted into the brain using a stereotaxic frame. The characterization of in vivo signals was assessed by electrochemical and pharmacological verification. Results support our experimental conditions that the measured oxidation current reflects variations in the .NO concentration in brain extracellular space. We report results from recordings in hippocampus and striatum upon stimulation of N-methyl-d-aspartate-subtype glutamate receptors. Moreover, the kinetics of .NO disappearance in vivo following pressure ejection of a .NO solution is also addressed.

• Microscopic technique for the detection of nitric oxide-dependent angiogenesis in an animal model.

  Authors: Seung Namkoong, Byoung-Hee Chung, Kwon-Soo Ha, Hansoo Lee, Young-Guen Kwon, Young-Myeong Kim

  Methods in enzymology. 441:393-402.

  Nitric oxide (NO) plays an important role in maintaining vascular homeostasis. The importance of NO in the vasculature is demonstrated by several experimental conditions, such as vascular endothelialNitric oxide (NO) plays an important role in maintaining vascular homeostasis. The importance of NO in the vasculature is demonstrated by several experimental conditions, such as vascular endothelial growth factor (VEGF)-induced angiogenesis. Thus, the NO metabolic pathway in endothelial cells could be one of the main contributing factors for angiogenesis. Although several methods have been used for measuring in vitro angiogenesis, a proper technique has not been developed for identifying in vivo NO-dependent angiogenesis. This chapter provides a new intravital microscopic method for detecting and measuring NO-dependent angiogenesis in a mouse model. This technique showed strong abdominal neovascularization in wild-type mice, but not eNOS knockout mice, locally injected with VEGF, as well as stimulation of angiogenesis in NO donor-injected mice. This technique also revealed the inhibitory effect of the NOS inhibitor N(G)-iminoethyl-L-ornithine in VEGF-mediated in vivo angiogenesis. This chapter describes intravital microscopy as a new imaging technique for detecting NO-dependent angiogenesis in an animal model.

• Chapter 15 analysis of neuronal cell death in mammals.

  Authors: Marcello D'Amelio, Virve Cavallucci, Adamo Diamantini, Francesco Cecconi

  Methods in enzymology. 446:259-76.

  Apoptosis, often defined as programmed cell death, plays a very important role in many physiologic and pathologic conditions. Therefore, detecting apoptotic cells or monitoring the cells progressingApoptosis, often defined as programmed cell death, plays a very important role in many physiologic and pathologic conditions. Therefore, detecting apoptotic cells or monitoring the cells progressing to apoptosis is an essential step in basic and/or applied research. Apoptosis is characterized by many biologic and morphologic changes of cells, for example, cytochrome c release from mitochondria, activation of caspases, DNA fragmentation, membrane blebbing, and formation of apoptotic bodies. On the basis of these changes, various assays have been designed to detect or quantify apoptotic cells. The goal of this chapter is to provide readers with a scientific guide to proven methods that highlight the current strategies for detecting apoptosis in the nervous system.

• Chapter 17 quantification of vascular endothelial cell apoptosis in vivo.

  Authors: Xuefeng Zhang, Sareh Parangi

  Methods in enzymology. 446:287-92.

  Regulation of vascular endothelial cell survival and apoptosis plays a crucial role during development and numerous physiologic and pathologic processes. Analyzing endothelial apoptosis in vivo isRegulation of vascular endothelial cell survival and apoptosis plays a crucial role during development and numerous physiologic and pathologic processes. Analyzing endothelial apoptosis in vivo is necessary not only for the understanding of many physiologic and pathologic processes but also for evaluating treatments that induce endothelial cell apoptosis. This chapter describes one of the more widely used protocols for detecting and quantifying vascular endothelial cell apoptosis by double-staining vascular endothelial cells with immunofluorescence and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL).

• Chapter 19 analysis of tnf-related apoptosis-inducing ligand in vivo through bone marrow transduction and transplantation.

  Authors: Keli Song, Raj Mariappan, Roya Khosravi-Far

  Methods in enzymology. 446:315-31.

  Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines. TRAIL has gained much attention because of its ability to preferentially kill tumorTumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines. TRAIL has gained much attention because of its ability to preferentially kill tumor cells with no apparent toxic side effects. Recently, different TRAIL receptor agonists, including TRAIL itself and various agonistic monoclonal antibodies against the two apoptosis-inducing human TRAIL receptors, have been developed as novel cancer therapeutics and are currently under investigation in clinical trials. However, the mechanisms by which TRAIL mediates its selective antineoplastic activity are still not well understood. In addition to playing a role in cancer immune surveillance and tumor suppression, TRAIL has been associated with immune homeostasis, inflammatory diseases, and autoimmunity. In light of the multifunctional role of TRAIL in mediating various pathologic conditions and the potential benefits of TRAIL-based therapies, the study of the physiologic significance of TRAIL is of great importance. Here, we describe a syngeneic system for the characterization of the in vivo function of TRAIL. By use of this model, in which the full-length murine TRAIL protein is overexpressed in the hematopoietic cells of wild-type mice, the in vivo tumoricidal activity of TRAIL overexpression can be studied on syngeneic murine tumor cell challenge, and the potential toxicity of TRAIL protein to normal tissues can also be analyzed.

• Chapter 21 caspase assays: identifying caspase activity and substrates in vitro and in vivo.

  Authors: Cristina Pop, Guy S Salvesen, Fiona L Scott

  Methods in enzymology. 446:351-67.

  The measurement of general caspase activity and the quantification of purified recombinant caspases in vitro can be accomplished with relative ease. But the determination of which caspases are activeThe measurement of general caspase activity and the quantification of purified recombinant caspases in vitro can be accomplished with relative ease. But the determination of which caspases are active in a cellular context is much more challenging. This is because commercially available small molecule substrates and inhibitors do not display sufficient specificity to dissect the complex interplay of caspase pathways. Here we describe procedures that can be used to validate which caspases are active in cell culture models and determine which caspases are responsible for specific cleavage events. We also recommend methods for working with recombinant initiator caspases in vitro and suggest ways to accurately assess the cleavage efficiency of natural caspase substrates.

• Chapter 23 Dissection of the BCL-2 Family Signaling Network with Stabilized alpha-Helices of BCL-2 Domains.

  Authors: Kenneth Pitter, Federico Bernal, James Labelle, Loren D Walensky

  Methods in enzymology. 446:387-408.

  The BCL-2 family of apoptotic proteins regulates the critical balance between cellular life and death and, thus, has become the focus of intensive basic science inquiry and a fundamental target forThe BCL-2 family of apoptotic proteins regulates the critical balance between cellular life and death and, thus, has become the focus of intensive basic science inquiry and a fundamental target for therapeutic development in oncology and other diseases. Classified based on the presence of conserved alpha-helical motifs and pro- and anti-apoptotic functionalities, BCL-2 proteins participate in a complex interaction network that determines cellular fate. The identification of BCL-2 homology domain 3 (BH3) as a critical death helix that engages and regulates BCL-2 family proteins has inspired the development of molecular tools to decode and drug the interaction network. Stabilized Alpha-Helices of BCL-2 domains (SAHBs) are structurally reinforced, protease-resistant, and cell-permeable compounds that retain the specificity of native BH3 death ligands and, therefore, serve as ideal reagents to dissect BCL-2 family interactions in vitro and in vivo. Here, we describe the in vitro and cell-based methods that exploit SAHB compounds to determine the functional consequences of BH3 interactions in regulating apoptosis.

• Chapter 1 analysis of autophagic and necrotic cell death in dictyostelium.

  Authors: Corinne Giusti, Artemis Kosta, David Lam, Emilie Tresse, Marie-Françoise Luciani, Pierre Golstein

  Methods in enzymology. 446:1-15.

  Non-apoptotic cell death types can be conveniently studied in Dictyostelium discoideum, an exceptionally favorable model not only because of its well-known genetic and experimental advantages, butNon-apoptotic cell death types can be conveniently studied in Dictyostelium discoideum, an exceptionally favorable model not only because of its well-known genetic and experimental advantages, but also because in Dictyostelium there is no apoptosis machinery that could interfere with non-apoptotic cell death. We show here how to conveniently demonstrate, assess, and study these non-apoptotic cell death types. These can be generated by use of modifications of the monolayer technique of Rob Kay et al., and either wild-type HMX44A Dictyostelium cells, leading to autophagic cell death, or the corresponding atg1(-) autophagy gene mutant cells, leading to necrotic cell death. Methods to follow these non-apoptotic cell death types qualitatively and quantitatively will be reported.

• Chapter 3 in vivo and in vitro methods for studying apoptotic cell engulfment in Drosophila.

  Authors: Elizabeth A Silva, Jemima Burden, Nathalie C Franc

  Methods in enzymology. 446:39-59.

  Proper development of all multicellular organisms involves programmed apoptosis. Completion of this process requires removal of the resulting cell corpses through phagocytosis by their neighbors orProper development of all multicellular organisms involves programmed apoptosis. Completion of this process requires removal of the resulting cell corpses through phagocytosis by their neighbors or by macrophages. Studies in C. elegans have been fruitful in the genetic dissection of key pathways, but they lack the professional immune system of higher organisms. Mammalian studies have identified a plethora of factors that are required for engulfment, but redundancy in the pathways has made it difficult to explain the genetic hierarchy of these factors. Thus, Drosophila has proven to be a useful evolutionary intermediate in which to examine this phenomenon. Here we describe methods used for dissecting the mechanisms and pathways involved in the engulfment of apoptotic cells by Drosophila phagocytes. Included are methods to be used for in vivo studies in the early embryo that can be used to examine engulfment of dying cells at various stages of embryogenesis. We also describe in vitro techniques for the use of Drosophila cell culture, including cell engulfment assays, that can be used for general phenotypic analysis, as well as live cell studies. We provide advice on imaging, including the preparation of samples for high-resolution microscopy and quantification of potential engulfment phenotypes for both in vivo and in vitro methods.

• Chapter 5 immortalized mouse epithelial cell models to study the role of apoptosis in cancer.

  Authors: Robin Mathew, Kurt Degenhardt, Liti Haramaty, Cristina M Karp, Eileen White

  Methods in enzymology. 446:77-106.

  Human cancer cell lines are widely used to model cancer but also have serious limitations. As an alternate approach, we have developed immortalized mouse epithelial cell model systems that areHuman cancer cell lines are widely used to model cancer but also have serious limitations. As an alternate approach, we have developed immortalized mouse epithelial cell model systems that are applicable to different tissue types and involve generation of immortalized cell lines that are genetically defined. By applying these model systems to mutant mice, we have extended the powerful approach of mouse genetics to in vitro analysis. By use of this model we have generated immortal epithelial cells that are either competent or deficient for apoptosis by different gain- and loss-of-function mutations that have revealed important mechanisms of tumor progression and treatment resistance. Furthermore, we have derived immortalized, isogenic mouse kidney, mammary, prostate, and ovarian epithelial cell lines to address the issues of tissue specificity. One of the major advantages of these immortalized mouse epithelial cell lines is the ability to perform biochemical analysis, screening, and further genetic manipulations. Moreover, the ability to generate tumor allografts in mice allows the integration of in vitro and in vivo approaches to delineate the mechanistic aspects of tumorigenesis. These model systems can be used effectively to determine the molecular requirements of epithelial tumorigenesis and tumor-promoting functions. This approach provides an efficient way to study the role of apoptosis in cancer and also enables the interrogation and identification of potential chemotherapeutic targets involving this pathway. Applying this technology to other mouse models can provide insight into additional aspects of oncogenesis.

• Chapter 7 phorbol ester-induced apoptosis and senescence in cancer cell models.

  Authors: Liqing Xiao, M Cecilia Caino, Vivian A von Burstin, Jose L Oliva, Marcelo G Kazanietz

  Methods in enzymology. 446:123-39.

  Protein kinase C (PKC) isozymes catalyze the phosphorylation of substrates that play key roles in the control in proliferation, differentiation, and survival. Treatment of cells with phorbol esters,Protein kinase C (PKC) isozymes catalyze the phosphorylation of substrates that play key roles in the control in proliferation, differentiation, and survival. Treatment of cells with phorbol esters, activators of classical and novel PKC isozymes, leads to a plethora of responses in a strict cell-type-dependent specific manner. Interestingly, a few cell models undergo apoptosis in response to phorbol ester stimulation, including androgen-dependent prostate cancer cells. This effect involves the autocrine secretion of death factors and activation of the extrinsic apoptotic cascade. We have recently found that in other models, such as lung cancer cells, phorbol esters lead to irreversible growth arrest and senescence. This chapter describes the methods we use to assess these phorbol ester responses in cancer cell models, focusing on apoptosis and senescence.

• Chapter 9 Studying p53-Dependent Cell Death In Vitro and In Vivo.

  Authors: Kageaki Kuribayashi, Niklas Finnberg, Wafik S El-Deiry

  Methods in enzymology. 446:159-73.

• Chapter 11 purification and bioassay of hedgehog ligands for the study of cell death and survival.

  Authors: Pilar Martinez-Chinchilla, Natalia A Riobo

  Methods in enzymology. 446:189-204.

  The Hedgehog (Hh) family of secreted ligands-composed of Sonic Hedgehog (Shh), Indian Hedgehog (Ihh), and Desert Hedgehog (Dhh)-possesses many roles during embryonic development, adult homeostasis,The Hedgehog (Hh) family of secreted ligands-composed of Sonic Hedgehog (Shh), Indian Hedgehog (Ihh), and Desert Hedgehog (Dhh)-possesses many roles during embryonic development, adult homeostasis, and cancer. The specific functions of the Hh proteins are intertwined with their requirement as survival factors in Hh-responsive cells. However, studies designed to dissect the anti-apoptotic role of Hhs have been hindered by the lack of simple approaches to purify large quantities of recombinant ligands in the average laboratory setting because of the natural modifications of these proteins with palmitic acid and cholesterol. In this chapter, we provide a comprehensive protocol for the expression of Shh, Ihh, and Dhh in Escherichia coli as fusion proteins with calmodulin-binding peptide to allow easy and rapid purification. The ligands are engineered with a new N-terminus containing two isoleucine residues to provide an essential hydrophobic interphase for achieving high biologic activity. The protocol includes a detailed description of a method for determination of the specific activity of the generated proteins by use of a cell culture-based luciferase approach.

• Chapter 13 ubiquitination mediated by inhibitor of apoptosis proteins.

  Authors: Sun-Mi Park, Shimin Hu, Tae H Lee, Xiaolu Yang

  Methods in enzymology. 446:225-35.

  Inhibitor of apoptosis (IAP) proteins are a family of evolutionarily conserved proteins that regulate apoptosis as well as other cellular processes. The functions of many IAPs are defined by theirInhibitor of apoptosis (IAP) proteins are a family of evolutionarily conserved proteins that regulate apoptosis as well as other cellular processes. The functions of many IAPs are defined by their RING domains, which possess E3 ubiquitin ligase activity and promote proteasomal degradation of an increasing number of target proteins. In this chapter, we describe the methods used in our laboratories to study the IAP's E3 activity.

• Globins and other nitric oxide-reative proteins, Part A. Preface.

  Authors: Robert K Poole

  Methods in enzymology. 436:xxiii-xxiv.

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