Apoptosis and Cell Proliferation, страница 4

Formalin grade.Antigen retrieval:Prepare 500 ml of 10 mM citric acid buffer. Incubate in a microwave oven at750 W until boiling. Place slides into the heated citric acid solution. Incubateonce more at 750 W. When solution is boiling, turn setting of microwaveoven to “keep warm” (about 100 W). Incubate for 15 min. Cool the slidesdown (for 5 min, at RT)Typical results: See following figures.Rinse three times in PBS; incubate 2 min in a separate jar of PBSBlock with PBS + 1% BSA (for 10 min, at RT)Remove blocking solution.

Add M30 working solution (1 h, RT)Wash slides three times in PBSCover with Anti-Mouse-Biotin1 (for 30 min, at RT)Wash slides three times in PBSCover with Streptavidin-POD2 (for 30 min, at RT)G Figure a: Detection of apoptosis in HeLa cells, treated with TNFand actinomycin D, using M30 CytoDEATH. Secondary detection withAnti-Mouse-Fluorescein and propidium iodide.Wash slides three times in PBSIncubate slides in a freshly prepared substrate solution (DAB or AEC at RT)until a clearly visible color developsCounterstain with hematoxilin, and mount the sectionAnalyze by a light microscopeG Figure b: Detection of apoptosis in human colon using M30 CytoDEATH(blue filter). Secondary detection with Anti-Mouse-Biotin, Streptavidin-PODand AEC as substrate, counterstained with hematoxilin.G Figure c: Detection of apoptosis in HeLa cells, using M30 CytoDEATH.Secondary detection with Anti-Mouse-Fluorescein.

Blue: untreated control cells.Red: Cells treated with TNF and actinomycin D.IXTrademarksABTS®is a registered trademark of Boehringer Mannheim GmbH, Mannheim, GermanyAlexa®is a trademark of Molecular Probes, Inc., Eugene, OR, USABOBO® is a trademark of Molecular Probes, Inc., Eugene, OR, USAX11.1Introduction21.1.11.1.21.1.3Terminology of cell deathDifferences between necrosis and apoptosisApoptotic Pathways2341.2Apoptosis Assay Methods61.2.11.2.1.11.2.1.21.2.1.31.2.21.2.2.11.2.2.21.2.2.31.2.2.41.2.3Methods for studying apoptosis in cell populationsAssays that measure DNA fragmentationAssays that measure apoptosis-induced proteases (caspases)Summary of methods for studying apoptosis in cell populationsMethods for studying apoptosis in individual cellsThe TUNEL enzymatic labeling assayAssays that measure membrane alterationsAssays that use DNA stainsSummary of methods for studying apoptosis in individual cellsDetection of apoptosis-related proteins8816222424313638401.3Cytotoxicity Assay Methods1.3.11.3.21.3.2.11.3.2.21.3.2.3Relationship between cytotoxicity, apoptosis and necrosisMethods for studying cytotoxicityAssays that measure plasma membrane leakageAssays that measure metabolic activitySummary of methods for studying cytotoxicity505050515860Chapter 1Cell Death –Apoptosis and NecrosisCell Death – Apoptosis and NecrosisIntroductionTerminology of cell death1.1 Introduction1.1.1 Terminology of cell deathCell death can occur by either of twodistinct1, 2 mechanisms, necrosis or apoptosis.

In addition, certain chemical compounds and cells are said to be cytotoxic tothe cell, that is, to cause its death.Someone new to the field might ask, what’sthe difference between these terms? Toclear up any possible confusion, we startwith some basic definitions.1Necrosis and apoptosisThe two mechanisms of cell death maybriefly be defined:Figure 1: Illustration of themorphological features of necrosisand apoptosis.Necrosis (“accidental” cell death) is thepathological process which occurs whencells are exposed to a serious physical orchemical insult.Apoptosis (“normal” or “programmed”cell death) is the physiological process bywhich unwanted or useless cells are eliminated during development and other normal biological processes.CytotoxicityCytotoxicity is the cell-killing property ofa chemical compound (such as a food, cosmetic, or pharmaceutical) or a mediator cell(cytotoxic T cell).

In contrast to necrosisand apoptosis, the term cytotoxicity doesnot indicate a specific cellular death mechanism.For example, cell-mediated cytotoxicity(that is, cell death mediated by either cytotoxic T lymphocytes [CTL] or natural killer [NK] cells) combines some aspects ofboth necrosis and apoptosis3, 4.HNecrosismitochondrial morphologicalchangesmembrane breakdownchromatin pattern conservednormalreversible swellingirreversible swellingdisintegrationApoptosismitochondrial morphologypreservedintact membranesDNAfragmentsnuclear changesnormal2condensation (cell blebbing)apoptotic bodiesfragmentationsecondary necrosis1.1.2 Differences between necrosisand apoptosisThere are many observable morphological(Figure 1, Table 1) and biochemical differences (Table 1) between necrosis and apoptosis2.Necrosis occurs when cells are exposed toextreme variance from physiological conditions (e.g., hypothermia, hypoxia) whichmay result in damage to the plasma membrane.

Under physiological conditions direct damage to the plasma membrane isevoked by agents like complement and lytic viruses.Necrosis begins with an impairment of thecell’s ability to maintain homeostasis, leading to an influx of water and extracellularions. Intracellular organelles, most notablythe mitochondria, and the entire cell swelland rupture (cell lysis).

Due to the ultimatebreakdown of the plasma membrane, thecytoplasmic contents including lysosomalenzymes are released into the extracellularfluid. Therefore, in vivo, necrotic cell deathis often associated with extensive tissuedamage resulting in an intense inflammatory response5.Apoptosis, in contrast, is a mode of celldeath that occurs under normal physiological conditions and the cell is an active participant in its own demise (“cellular suicide”). It is most often found during normal cell turnover and tissue homeostasis,embryogenesis, induction and maintenanceof immune tolerance, development of thenervous system and endocrine-dependenttissue atrophy.Cells undergoing apoptosis show characteristic morphological and biochemical features.6 These features include chromatinaggregation, nuclear and cytoplasmic condensation, partition of cytoplasm and nucleus into membrane bound-vesicles (apoptotic bodies) which contain ribosomes,morphologically intact mitochondria andnuclear material.

In vivo, these apoptoticbodies are rapidly recognized and phagocytized by either macrophages or adjacentepithelial cells.7 Due to this efficient mechanism for the removal of apoptotic cellsin vivo no inflammatory response is elicited. In vitro, the apoptotic bodies as wellas the remaining cell fragments ultimatelyswell and finally lyse. This terminal phaseof in vitro cell death has been termed “secondary necrosis” (Figure 1).NecrosisMorphological featuresP Loss of membrane integrity1ApoptosisP Membrane blebbing, but no loss of integrityP Aggregation of chromatin at the nuclear membraneP Begins with swelling of cytoplasm and mitochondriaP Begins with shrinking of cytoplasm and condensation of nucleusP Ends with total cell lysisP Ends with fragmentation of cell into smaller bodiesP No vesicle formation, complete lysisP Formation of membrane bound vesicles (apoptotic bodies)P Disintegration (swelling) of organellesP Mitochondria become leaky due to pore formation involving proteins of thebcl-2 family.Biochemical featuresP Loss of regulation of ion homeostasisP No energy requirement (passive process, also occurs at 4°C)P Tightly regulated process involving activation and enzymatic stepsP Energy (ATP)-dependent (active process, does not occur at 4°C)P Random digestion of DNA (smear of DNA after agarose gel electrophoresis) P Non-random mono- and oligonucleosomal length fragmentation of DNA(Ladder pattern after agarose gel electrophoresis)P Postlytic DNA fragmentation (= late event of death)Physiological significanceP Affects groups of contiguous cellsP Evoked by non-physiological disturbances (complement attack, lyticviruses, hypothermia, hypoxia, ischemica, metabolic poisons)P Phagocytosis by macrophagesP Significant inflammatory responseG Table 1: Differential features and significance of necrosis and apoptosis.Cell Death – Apoptosis and NecrosisIntroductionDifferences between necrosis and apoptosisP Prelytic DNA fragmentationP Release of various factors (cytochrome C, AIF) into cytoplasm bymitochondriaP Activation of caspase cascadeP Alterations in membrane asymmetry (i.e., translocation of phosphatidylserine from the cytoplasmic to the extracellular side of the membrane)P Affects individual cellsP Induced by physiological stimuli (lack of growth factors, changes inhormonal environment)P Phagocytosis by adjacent cells or macrophagesP No inflammatory response3Cell Death – Apoptosis and NecrosisIntroductionApoptotic Pathways1.1.3 Apoptotic PathwaysScientists now recognize that most, if notall, physiological cell death occurs by apoptosis, and that alteration of apoptosis mayresult in a variety of malignant disorders.Consequently, in the last few years, interestin apoptosis has increased greatly.

Greatprogress has been made in the understanding of the basic mechanisms of apoptosisand the gene products involved (Figure 2below, Table 25, see Appendix, page 115).1G Figure 2: Apoptotic pathways. This apoptotic pathways chart represents a compendium of information on different cell lines, from various sources.As the dynamic field of apoptosis changes, the information shown here will likely change.

Table 25 in the Appendix, page 115 contains a list of sources that canbe consulted for more information about the items on this chart.Note: Request a wall chart of these pathways by filling out the reply card in the back of the manual.For additional information on the apoptotic pathways, please visit us on the Internet athttp://biochem.boehringer-mannheim.com/techserv/apoptosis/index.htm4Key elements of the apoptotic pathwayinclude:Death receptorsApoptosis has been found to be induced viathe stimulation of several different cell surface receptors in association with caspaseactivation. For example, the CD95 (APO-1,Fas) receptor ligand system is a criticalmediator of several physiological andpathophysiological processes, includinghomeostasis of the peripheral lymphoidcompartment and CTL-mediated targetcell killing. Upon cross-linking by ligandor agonist antibody, the Fas receptor initiates a signal transduction cascade whichleads to caspase-dependent programmedcell death.Membrane alterationsIn the early stages of apoptosis, changesoccur at the cell surface and plasma membrane.