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Conversely,if a rat is treated with the drug phenobarbital-whichstimulates liver cell division (and thereby liver enlargement)-and then the phenobarbital treatment is stopped, apoptosis in the livergreatly increasesuntil the liver has returned to its original size, usually within aweek or so.
Thus, the liver is kept at a constant size through the regulation ofboth the cell death rate and the cell birth rate, although the control mechanismsresponsible for such regulation are largely unknor,rm.Apoptosis occurs at a staggeringlyhigh rate in the adult human bone marrow where most blood cells are produced. Here, for example, neutrophils (a typeof white blood cell discussed in Chapter 23) are produced continuously in verylarge numbers, but the vast majority die by apoptosis in the bone marrow withina few days without ever functioning. This apparently futile cycle of productionand destruction serves to maintain a ready supply of short-lived neutrophilsthat can be rapidly mobilized to fight infection wherever it occurs in the body.Compared with the life of the organism, cells are evidently cheap.Animal cells can recognize damage in their various organelles and, if thedamage is great enough, they can kill themselves by undergoing apoptosis.
Animportant example is DNA damage, which can produce cancer-promotingmutations if not repaired. Cells have various ways of detecting DNA damage,and, if they cannot repair it, they often kill themselves by undergoing apoptosis.ApoptoticCellsAre BiochemicallyRecognizableCells undergoing apoptosis not only have a characteristic morphology but alsodisplay characteristic biochemical changes,which can be used to identify apoptotic cells. During apoptosis, for example, an endonucleasecleavesthe chromosomal DNA into fragments of distinctive sizes;because the cleavagesoccur inthe linker regions between nucleosomes, the fragments separateinto a characteristic ladder pattern when analyzed by gel electrophoresis (Figure 18-44).Moreover, the cleavage of DNA generates many new DNA ends, which can bemarked in apoptotic nuclei by using a labeled nucleotide in the so-calledTUNELtechnique (Figure l8-4B).An especially important change occurs in the plasma membrane of apoptotic cells.The negatively charged phospholipid phosphatidylserineis normallyexclusively located in the inner leaflet of the lipid bilayer of the plasma membrane (seeFigures 10-3 and 10-16), but it flips to the outer leaflet in apoptoticcells,where it can serve as a marker of these cells.The phosphatidylserine on thesurface of apoptotic cells can be visualized with a labeled form of the Annexin Vprotein, which specifically binds to this phospholipid.
The cell-surface phosphatidylserine is more than a convenient marker of apoptosis for biologists; ithelps signal to neighboring cells and macrophages to phagocytose the dyingcell. In addition to serving as an "eat me" signal, it also blocks the inflammationoften associatedwith phagocltosis: the phosphatidylserine-dependent engulfment of apoptotic cells inhibits the production of inflammation-inducing signalproteins (cltokines) by the phagocytic cell.Macrophages will phagoc)'tose most types of small particles, including oildroplets and glass beads, but they do not phagocytose any healthy cells in theanimal, presumably because healthy cells express"don't eat me" signal moleculeson their surface.Thus, in addition to expressingcell-surface"eat me" signalssuchas phosphatidylserine that stimulate phagocltosis, apoptotic cells must lose orinactivate their "dont eat me" signals in order for macrophagesto ingest them.1118Chapter18:ApoptosisFigure18-4 Markersof apoptosis.(A)Cleavageof nuclearDNAinto acharacteristicladderpatternof fragments.Mousethymuslymphocytesweretreatedwith an antibodyagainstthe cell-surfacedeathreceptorFas(discussedlater),inducingthe cellsto undergoapoptosis.Aftervarioustimes(indicatedin hoursat the top of the figure),DNAwasextracted,andthe fragmentswere separatedby sizeby electrophoresisin an agarosegeland stainedwith ethidiumbromide.(B)TheTUNELtechniquewasusedtolabelthe cut endsof DNAfragmentsin the nucleiof apoptoticcellsin atissuesectionofa developingchickleg bud;this crosssectionthroughtheskinand underlyingtissueis from a regionbetweentwo developingdigits,as indicatedin the underlyingdrawing.The procedureis calledthe TUNELdUTPnickend labeling)techniquebecausethe enzymefidT-mediated(TdT)addschainsof labeledterminaldeoxynucleotidyltransferase(dUTP)deoxynucleotideto the 3aOHendsof DNAfragments.(A,fromD.
Mcllroyet al.,GenesDev.14:549-558,2000.With permisisonfrom ColdSpringHarborLaboratoryPress;B,from V.Zuzarte-Luisand J.M.Hu116,lnt.J. Dev.Biol.46:871-876,2002.With permissionfrom UBCPress.)time (hr)0Cells undergoing apoptosis often lose the electrical potential that normallyexists across the inner membrane of their mitochondria (discussed in chapterl4). This membrane potential can be measured by the use of positively chargedfluorescent dyes that accumulate in mitochondria,driven by the negative chargeon the inside of the inner membrane.
A decrease in the labeling of mitochondriawith these dyes helps to identify cells that are undergoing apoptosis. As we discuss later, proteins such as cytochrome c are usually released from the spacebetween the inner and outer membrane (the intermembranespace) of mitochondria during apoptosis, and the relocation of cytochrome c from mitochondria to the cytosol can be used as another marker of apoptosis (see Figure I8-7).ApoptosisDependson an IntracellularProteolyticCascadeThatlsMediatedby CaspasesThe intracellular machinery responsible for apoptosis is similar in all animalcells. It depends on a family of proteasesthat have a cysteine at their active siteand cleave their target proteins at specific aspartic acids. They are thereforecalled caspases (c for cysteine and asp for aspartic acid).
caspases are synthesized in the cell as inactive precursors, or procaspases,which are typically activated by proteolytic cleavage.Procaspasecleavageoccurs at one or two specificaspartic acids and is catalyzedby other (alreadyactive) caspases;the procaspaseis split into a large and a small subunit that form a heterodimer, and two suchdimers assemble to form the active tetramer (Figure l8-5A). once activated,caspasescleave, and thereby activate, other procaspases,resulting in an amplifying proteolytic cascade (Figure l8-58).Not all caspasesmediate apoptosis. Indeed, the first caspaseidentified wasa human protein calred interleukin-l-conuerting enzyme (ICE), which is concerned with inflammatory responses rather than with cell death; ICE cuts outthe inflammation-inducing cltokine interleukin-I eLl) from a larger precursorAs shor,rmin Figure 18-58 and rable l8-1, some of the procaspases thatoperate in apoptosis act at the start of the proteolytic cascadeand are called initiator procaspases; when activated, they cleave and activate dor,tmstreamexecutioner procaspases, which, then cleave and activate other executioner procaspases' as well as specific target proteins in the cell.
Among the many targetproteins cleaved by executioner caspasesare the nuclear lamins (see Figure18-58), the cleavageof which causes the irreversible breakdown of the nuclearlamina (discussedin chapter 16).Another target is a protein that normally holds(8)APOPTOSIs1119(A) procaspaseactivationD yc l e a v a g eactivecaspase( B ) c a s p a s ce a s c a d eo n e m o l e c u l eo factive initiator caspaseinactiveprocaspasesactrvecaspaseprodomains/lt ttt\/// |\\\/ti |\r\gf executiener'caglseflnahymolecules/\nn/\/\I'.acleavage ofcytosolicproteinthe DNA-degrading enzyme mentioned earlier (an endonuclease)in an inactiveform; its cleavagefrees the endonuclease to cut up the DNA in the cell nucleus.Other target proteins include components of the cltoskeleton and cell-celladhesion proteins that attach cells to their neighbors; the cleavageof these proteins helps the apoptotic cell to round up and detach from its neighbors, making it easierfor a healthy neighboring cell to engulf it, or, in the caseof an epithelial cell, for the neighbors to extrude the apoptotic cell from the cell sheet.
Thecaspasecascadeis not only destructive and self-amplifying but also irreversible,so that once a cell reaches a critical point along the path to destruction, it cannot turn back.The caspasesrequired for apoptosis vary depending on the cell type andstimulus. Inactivation of the mouse gene encoding caspase-3,an executionercaspase,for example, reduces normal apoptosis in the developing brain. As aresult, the mouse often dies around birth with a deformed brain that containstoo many cells. Apoptosis occurs normally, however, in many other organs ofsuch mice.From the earliest stagesof an animal's development, healthy cells continuously make the procaspasesand other proteins required for apoptosis.Thus, theapoptosis machinery is always in place; all that is needed is a trigger to activateit. How then, is a caspasecascadeinitiated? In particular, how is the first procaspasein the cascadeactivated?Initiator procaspaseshave a long prodomain,which contains a caspase recruitment domain (CARD) that enables them toassemblewith adaptor proteins into actiuation complexeswhen the cell receivesa signal to undergo apoptosis.
Once incorporated into such a complex, the initiator procaspasesare brought into close proximity, which is sufficient to activate them; they then cleave each other to make the processirreversible.The activated initiator caspases then cleave and activate executioner procaspases,thereby initiating a proteolytic caspasecascade,which amplifies the death signal and spreads it throughout the cell.The two best understood signaling pathways that can activate a caspasecascade leading to apoptosis in mammalian cells are called the extrinsic pathwayand the intrinsic pathway.
Each uses its own initiator procaspasesand activation complex, as we now discuss.Table18-1 SomeHumanCaspasesCaspasesinvolvedin inflammationCaspasesinvolvedin apoptosisInitiatorcaspasesExecutionercaspases1 (lCE),4,5caspasesc a s p a s 2e ,s8 , 9 , 1 0caspases3,6, 7cleavage ofnuclear laminFigure 18-5 Procaspaseactivationisduring apoptosis.(A)Eachcaspaseinitiallymadeas an inactiveproenzyme(procaspase).areSomeprocaspasesactivatedby proteolyticcleavageby antwo cleavedfragmentsactivatedcaspase:moleculesfrom eachof two procaspaseassociateto form an activecaspase,whichis a tetramerof two smalland twolargesubunits;the prodomainsareas indicated.(B)Theusuallydiscarded,firstprocaspasesactivatedarecalledwhich then cleaveinitiatorprocaspases,and activate many executionerprocosposeproducingan amplifyingmolecules,chainreaction(a proteolyticcaspasecaspasesthencascade).Theexecutionercleavea varietyof key proteinsin the cell,includingspecificcytosolicproteinsandnuclearlamins,as shownhere,leadingtothe controlleddeathof the cell.Althougharenot shown,the initiatorprocaspasesactivatedby adaptorproteinsthat bringtogetherin closethe procaspasesproximitywithin an activationcomplex;cleavealthoughthe initiatorprocaspaseseachotherwithinthe complex,thecleavageservesonlyto stabilizetheactiveprotease.| 120Chapter18:Apoptosisk i l l e rl y m p h o c y t eFasligandFasdeath receptord e a t hd o m a i ndeath effectoractivatedc a s p a s e -o8r 1 0//l l\\+ACTIVATIONANDCLEAVAGEOF-10,PROCASPASE-8,OR BOTHCell-SurfaceDeathReceptorsActivatethe ExtrinsicPathwayofApoptosisExtracellular signal proteins binding to cell-surface death receptors trigger theextrinsic pathway of apoptosis.
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