Apoptosis and Cell Proliferation, страница 5

One of these plasma membrane alterations is the translocation of phosphatidylserine (PS) from the inner side of theplasma membrane to the outer layer, bywhich PS becomes exposed at the externalsurface of the cell.Protease cascadeSignals leading to the activation of a familyof intracellular cysteine proteases, the caspases, (Cysteinyl-aspartate-specific proteinases) play a pivotal role in the initiationand execution of apoptosis induced by various stimuli.

At least 11 different membersof caspases in mammalian cells have beenidentified. Among the best-characterizedcaspases is caspase-1 or ICE (Interleukin1b-Converting Enzyme), which was originally identified as a cysteine protease responsible for the processing of interleukin1b.Mitochondrial changesMitochondrial physiology is disrupted incells undergoing either apoptosis or necrosis. During apoptosis mitochondrial permeability is altered and apoptosis specificprotease activators are released from mitochondria. Specifically, the discontinuityof the outer mitochondrial membrane results in the redistribution of cytochrome Cto the cytosol followed by subsequentdepolarization of the inner mitochondrialmembrane.

Cytochrome C (Apaf-2) releasefurther promotes caspase activation bybinding to Apaf-1 and therefore activating Apaf-3 (caspase 9). AIF (apoptosis inducing factor), released in the cytoplasm,has proteolytic activity and is by itself sufficient to induce apoptosis.Cell Death – Apoptosis and NecrosisIntroductionApoptotic Pathways1DNA fragmentationThe biochemical hallmark of apoptosis isthe fragmentation of the genomic DNA, anirreversible event that commits the cell todie and occurs before changes in plasmamembrane permeability (prelytic DNAfragmentation).

In many systems, thisDNA fragmentation has been shown to result from activation of an endogenous Ca2+and Mg2+-dependent nuclear endonuclease. This enzyme selectively cleaves DNAat sites located between nucleosomal units(linker DNA) generating mono- and oligonucleosomal DNA fragments.Note: For more information about the elements of the pathways as well as synonymsand abbreviations, please see Table 25 inthe Appendix, page 115.5Cell Death – Apoptosis and NecrosisApoptosis Assay Methods1.2 Apoptosis Assay MethodsOriginally, to study both forms of celldeath, necrosis and apoptosis, cytotoxicityassays were used.

These assays were principally of two types:E Radioactive and non-radioactive assaysthat measure increases in plasma membrane permeability, since dying cells become leaky.1E Colorimetric assays that measure reduction in the metabolic activity of mitochondria; mitochondria in dead cellscannot metabolize dyes, while mitochondria in live cells can.Note: For a detailed discussion of both typesof cytotoxicity assay, see Section 1.3, beginning on page 50 of this guide.However, as more information on apoptosis became available, researchers realizedthat both types of cytotoxicity assays vastly underestimated the extent and timingof apoptosis.

For instance, early phases ofapoptosis do not affect membrane permeability, nor do they alter mitochondrialactivity. Although the cytotoxicity assays might be suitable for detecting the later stages of apoptosis, other assays wereneeded to detect the early events of apoptosis.In concert with increased understanding ofthe physiological events that occur duringapoptosis, a number of assay methods havebeen developed for its detection. For in-6stance, these assays can measure one of thefollowing apoptotic parameters:E Fragmentation of DNA in populationsof cells or in individual cells, in whichapoptotic DNA breaks into differentlength pieces.E Alterations in membrane asymmetry.Phosphatidylserine translocates fromthe cytoplasmic to the extracellular sideof the cell membrane.E Activation of apoptotic caspases.

Thisfamily of proteases sets off a cascade ofevents that disable a multitude of cellfunctions.E Release of cytochrome C and AIF intocytoplasm by mitochondria.For practical reasons, we have divided thischapter into two broad categories: assaysthat measure apoptosis in cell populations(Section 1.2.1 of this guide) and assays thatmeasure apoptosis in individual cells (Section 1.2.2 of this guide).For a discussion of the advantages and limitations of all types of apoptosis assays, readSections 1.2.1.3 and 1.2.2.4 of this guide.For discussions of particular assays, turn tothe pages indicated in the method selectionguide (Figure 3).StartCytotoxicityDetection Kit(LDH)(see page 52)Cellular DNAFragmentationELISA(see page 54)CytotoxicityAre you studyingnecrosis, apoptosis, or cytotoxicity?NecrosisAre you studyingcell populationsor individualcells?CellPopulationsIndividual cellsApoptosisAnnexin-VFLUOS StainingKit (see page 32)What techniquesdo you performin the lab?Immunosorbentenzyme assayELISACellPopulationAre you studyingcell populationsor individualcells?WesternblottingGelelectrophoresisIndividualcellsDo you analyzedata by FACS ormicroscopy?MicroscopyFACSCytotoxicityDetection Kit(LDH)(see page 52)Cellular DNAFragmentationELISA(see page 54)Cell DeathDetectionELISAPLUS(see page 13)1Do you usefluorescence orlight microscopy?LightFluorescenceCaspase 3Activity Assay(see page 17)Cell Death Detection ELISAPLUS(see page 13)Cellular DNAFragmentationELISA(see page 54)Anti-PARP(see page 20)Apoptotic DNALadder Kit(see page 11)In Situ Cell DeathDetection KitFluorescein(see page 27)Annexin-VFLUOS(see page 32)Annexin-VAlexa™ 568(see page 32)Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsIn Situ Cell DeathDetection Kit,POD(see page 29)In Situ Cell DeathDetection Kit, AP(see page 29)Annexin-V-Biotin(see page 34)G Figure 3: Method/Product selection guide.7Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsMethods for studying apoptosis in cell populations1.2.1 Methods for studying apoptosisin cell populationsA number of methods have now been developed to study apoptosis in cell populations.

We focus on two key apoptoticevents in the cell:11Apoptosis and cell mediated cytotoxicity are characterized by cleavage of thegenomic DNA into discrete fragmentsprior to membrane disintegration. Because DNA cleavage is a hallmark forapoptosis, assays which measure prelytic DNA fragmentation are especiallyattractive for the determination of apoptotic cell death. The DNA fragmentsmay be assayed in either of two ways:E As “ladders” (with the 180 bp multiples as “rungs” of the ladder) derivedfrom populations of cells, e.g., withthe Apoptotic DNA Ladder Kit (described on page 11 of this guide).E By quantification of histone complexed DNA fragments with anELISA (described on page 13 of thisguide).2Further, researchers discovered thatproteases were involved in the earlystages of apoptosis.

The appearance ofthese caspases sets off a cascade ofevents that disable a multitude of cellfunctions. Caspase activation can beanalyzed in different ways:E By an in vitro enzyme assay. Activityof a specific caspase, for instance caspase 3, can be determined in cellularlysates by capturing of the caspaseand measuring proteolytic cleavageof a suitable substrate (described onpage 17 of this guide).E By detection of cleavage of an in vivocaspase substrate.

For instance caspase 3 is activated during early stages(as shown in Figure 2). Its substratePARP (Poly-ADP-Ribose-Polymerase) and the cleaved fragments can bedetected with the anti PARP antibody (described on page 20 of thisguide).8If you’re just starting out in the field,however, it may be difficult to decide howbest to assay apoptosis in your system.Thus, in the following sections, we willdescribe details of each of these apoptosisassays.1.2.1.1 Assays that measure DNAfragmentationThe biochemical hallmark of apoptosis isthe fragmentation of the genomic DNA, anirreversible event that commits the cell todie.

In many systems, this DNA fragmentation has been shown to result from activation of an endogenous Ca2+ and Mg2+dependent nuclear endonuclease. This enzyme selectively cleaves DNA at sites located between nucleosomal units (linkerDNA) generating mono- and oligonucleosomal DNA fragments (Figure 4).These DNA fragments reveal, upon agarose gel electrophoresis, a distinctive ladderpattern consisting of multiples of an approximately 180 bp subunit8.Radioactive as well as non-radioactivemethods to detect and quantify DNA fragmentation in cell populations have beendeveloped.

In general, these methods arebased on the detection and/or quantification of either low molecular weight (LMW)DNA which is increased in apoptotic cellsor high molecular weight (HMW) DNAwhich is reduced in apoptotic cells (Figure 5). The underlying principle of thesemethods is that DNA, which has undergone extensive double-stranded fragmentation (LMW DNA) may easily be separatedfrom very large, chromosomal lengthDNA (HMW DNA), e.g., by centrifugation and filtration.“Beads-on-a-string”form of chromatin (HMW-DNA)Mono- and Oligonucleosomes(LMW-DNA)“DNA ladder”after gel electrophoresisHistone octamer ofnucleosome coreDistance between cuts =multiple of 180 base pairsEndonucleaseDNA subjected togel electrophoresisBase pairsLinker DNA1700520360180For the quantification of DNA fragmentation, most methods involve a step in whichthe DNA of the cells has to be labeled: Prior to the addition of the cell death-inducingagent or of the effector cells, the (target)cells are incubated either with the [3H]thymidine ([3H]-dT) isotope or the nucleotide analog 5-bromo-2’-deoxyuridine(BrdU).