Encyclopedia of Physical Science and Technology - Molecular Biology

110 Pages·2001·3.921 MB·English

by  Robert A. Meyers (Editor)

Table of Contents

(Subject Area: Molecular Biology)

Article Authors Pages in the Encyclopedia

Cell Death (Apoptosis) Masato Enari Pages 541-554
Chromatin Structure and Fyodor D. Urnov and
Pages 809-829
Modification Alan P. Wolffe
DNA Testing in Forensic
Moses S. Schanfield Pages 589-602
Science
Gene Expression, Regulation
Göran Akusjärvi Pages 501-516
of
K. Michael Pollard and
Immunology-Autoimmunity Pages 679-691
Eng M. Tan
Alessandra Poggi and
Ribozymes Pages 253-261
John J. Rossi
R. A. Cox and H. R. V.
Translation of RNA to Protein Pages 31-51
Arnstein
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Cell Death(Apoptosis)
Masato Enari
ImperialCollegeSchoolofMedicineatSt.Mary’s
I. Overview
II. DeathFactorsandTheirReceptors
III. ApoptoticProteases,Caspases
IV. SignalTransductionofDeath
Factor-MediatedApoptosis
V. Cell-FreeSysteminApoptosis
VI. ApoptoticDNase,CADandItsInhibitor,ICAD
VII. MolecularMechanismofCADandICAD
VIII. PhysiologicalDNAFragmentationand
PhagocytosisofApoptoticCells
IX. Perspectives
GLOSSARY cellsexpressingtheircorrespondingreceptorsortoat-
tenuatekillingactivity.
Apoptosis Thetypicalprocessinphysiologicalcelldeath DNases Enzymes possessing DNA-cleaving activity.
that is accompanied by nuclear and cytoplasmic con- SomeDNasesparticipateinchromosomalDNAdegra-
densation,fragmentationofcellbodies,chromosomal dationduringapoptosis.
DNA fragmentation, loss of mitochondrial function, DNAfragmentation ChromosomalDNAfromapoptotic
and alterations of cell membrane composition. It is cellsgivesrisetoaladderpatternonagarosegels,due
distinct in these regards from necrosis. The term was tomultimericnucleosomalunits(∼180basepairs).
createdbyWyllieandKerr. Death receptors Proteins belonging to TNF receptor
Caspases Cysteine proteases, some of which are acti- family that occur on cell surface, and mediate killing
vated during apoptosis. Some caspases are involved byeffectorcellsexpressingtheircognateligands.
inprocessingofcytokines. Intracellularsignaltransduction Thecellularmachin-
Cell-free system A biochemical technique to be recon- ery that mediates external signals including hor-
stitutedcellulareventsasinvitroreaction. mones, neurotransmitters, cell growth, differentiation
Death factors Proteins belonging to the tumor necrosis anddeathfactors,orstresstotheirultimatetargets.
factor (TNF) superfamily that occur on the cell sur- Phagocytosis Theprocessbywhichphagocytes,suchas
facesasmembrane-boundfactorsandintheextracel- macrophages and neutrophils, engulf useless and un-
lular compartment as soluble factors. They kill target necessarycells.
541
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542 CellDeath(Apoptosis)
APOPTOSIS,orprogramedcelldeath,playsanimpor- andKerrin1972.Inthenecroticprocess,swellingofcells
tantroleinthedevelopmentoforganismsandinthemain- precedes their explosion and results in the release of in-
tenanceofhomeostasis.Thefailureofapoptoticprograms tracellular components that may be toxic to other cells.
causes various diseases. So far, many genes regulating Inapoptosis,thedyingcellsexhibitnuclearandcytoplas-
apoptosishavebeenidentifiedandthemolecularmecha- miccondensation,fragmentationofcellbodies,chromo-
nismofapoptosisisbeingclarified.Inthisarticle,Iwill somal DNA fragmentation into nucleosomal units, loss
discusscelldeathelicitedthroughdeathreceptors. of mitochondrial function, and alterations of cell mem-
brane composition (Fig. 1). Subsequently, apoptotic cells
areengulfedbyphagocytesandneighboringcells,andare
I. OVERVIEW recycled.Mostcellssufferingphysiologicalcelldeathun-
dergotheapoptoticprocessandthesuperfluousorharmful
Homeostasisinmulticellularorganismsisbasedonabal- cellsgeneratedduringthedevelopmentalprocessarere-
ancebetweenlifeanddeathofcells.Apoptosiswasrecog- movedbyapoptosis.Forexample,apoptosisoccursintail
nizedasaphenomenondistinctfromnecrosisbyWyllie resorption,neuronalnetworkformation,clonaldeletionof
FIGURE1 (a)Fas-inducedapoptosisinlymphoidcells(WR19LcellsoverexpressingFas).Thecellswereincubated
with0.5µg/mlofanagonisticanti-Fasantibodyat37◦Cfor120minandtheirultrastructurewasexaminedunder
atransmissionelectronmicroscope.Theelectronmicrographofuntreatedcellsisshownintheupperpanel.Bars,
1µm.(b)ChromosomalDNAofgrowingcells(lane1)ordyingcells(lane2)wasrunthrougha1.5%agarosegel.M
indicatesmolecularweightmarkers.
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CellDeath(Apoptosis) 543
immatureandautoreactiveTcells,WolffianandMullerian a death domain (DD) that is required for transducing
ductregressionduringsexualdevelopment,tumorregres- apoptotic signals to cells. Six DD-containing death re-
sion, and in elimination of virus-infected cells. Further- ceptors, namely Fas/Apo1/CD95, type I TNF recep-
more,ithasbeensuggestedthatapoptosisoccursinmany tor (TNFRI), DR3/Apo3/WSL-1/TRAMP, DR4/TRAIL-
diseasessuchascancer,fulminanthepatitis,acquiredim- R1 (TNF-related apoptosis-inducing ligand receptor-1),
munedeficiencysyndrome(AIDS),diabetesmellitus,and DR5/TRAIL-R2/TRICK2/KILLER and DR6 have been
neurodegenerativedisorderssuchasAlzheimer’sdisease identifiedsofar.Inaddition,thereareDD-lessdeathre-
andpriondisease. ceptors such as type II TNF receptor (TNFRII), CD27,
Growthanddifferentiationofcellsarestrictlyregulated CD30,CD40,andlymphotoxin-βreceptor.Amongthese,
by factors such as cytokines and low-molecular-weight TNFRII,CD27,andCD30inducetheexpressionofboth
compounds such as steroid hormones. These factors are TNFandTNFRIbytheircognateligands,andsubsequent
generallyboundtothecorrespondingreceptorsinorderto inductionofapoptosisappearstooccur.
transducetheappropriatecellsignals,topromotegrowth On the other hand, death factors are a member of
anddifferentiation.Ontheotherhand,apoptoticcelldeath the TNF family and exist on the cell surface or as
is aggressively controlled by a number of polypeptides, soluble factors. Most death factors are synthesized as
so-calleddeathfactorsexposedatthecellsurfaceorcir- typeII-membraneproteinsandsubjectedtosheddingby
culatinginthebodyassolublefactorsinsomesituations. membrane-associatedmetalloproteinasesinordertogen-
Growthanddifferentiationfactorsactviatranscriptional erate soluble forms of death factors. A well-established
regulationthroughtheactivationofaseriesofproteinki- mechanismforsheddingofdeathfactorsisseeninthecase
nases.Ontheotherhand,deathfactorsexecuteapoptosis ofTNF.Membrane-boundTNF(memTNF)iscleavedat
throughtheactivationofcaspases,andmanyproteinses- theoutercellularmembranebyamembrane-spanningpro-
sentialforcellsurvivalaredegradedbytheseactivatedcas- tease,so-calledTNFα-convertingenzyme(TACE),which
pases.Itiscurrentlybelievedthatapoptoticdeathisdueto is a member of metalloproteinase-disintegrin (ADAM)
thedegradationofmanyfunctionalproteinsbycaspases. family. memTNF is superior to soluble TNF (sTNF) in
Cell-free systems for the study of apoptosis have been activating TNFRII in various cellular responses, includ-
establishedandfacilitateourunderstandingoftheunder- ing T-cell proliferation, inflammation, and cytotoxicity.
lyingmolecularmechanisms.Severalfactorsinvolvedin These results imply that memTNF regulates cellular re-
apoptoticpathwayshavebeenidentifiedandpartofthese sponsesviarestrictedcell-to-cellinteractionunderphysi-
pathways has been revealed by biochemical approaches ologicalconditionsandthatsTNFmayattenuateTNFRII-
based on cell-free apoptosis system. Regulatory mecha- mediated responses. The shedding mechanism for other
nisms for apoptosis include: the CED-3/caspase family deathfactorsmaybesimilartothatforTNF.Likeinshed-
proteasesandCED-4/Apaf-1familywhichactasexecu- ding of TNF, the membrane form of Fas ligand (mem-
tors of apoptosis; CED-9/Bcl-2 family (including anti- FasL) is also cleaved by an unknown metalloproteinase
apoptotic and pro-apoptotic factors) which act as regu- other than TACE present on the plasma membranes. In
lators of apoptosis, and many factors which contribute addition, the soluble form of Fas ligand (sFasL) inhibits
toapoptoticmorphologicalchangeshavebeenidentified. mFasL-mediated apoptosis in human peripheral blood T
Here, I shall focus on the currently proposed molecular lymphocytesinvitro.
mechanismsofdeathreceptoractivityandcaspaseactiva- It is believed that death receptors are activated by
tion.Moreover,IwillalsodiscusstheDNaseresponsible ligand-induced trimerization, as opposed to the activa-
for apoptotic DNA fragmentation, both in vitro and in tion of growth factor receptors, which occurs by dimer-
vivo,andfinallythemechanismbywhichapoptoticcells ization. Most death factors, but not all, are present as
arecleared. trimers.X-raystructuralanalyseshaverevealedthatTNF-
α,TNF-β,CD40L,andTRAILarehomotrimericproteins.
Amongthese,TRAILhasuniquecharacteristics.TRAIL
II. DEATH FACTORS AND requiresazincionforbiologicalactivityandselectively
THEIR RECEPTORS induces apoptosis in mouse tumor cells but not in nor-
malcells.Moreover,administrationofsolubleTRAILto
Many cells have death receptors on the surface of their mice implanted with human tumors causes effective re-
plasmamembranesandapoptosisistriggeredbytheircog- duction of tumor size without any injury of normal tis-
nate ligands. Death receptors belong to the superfamily sues.TheseresultssuggestthatTRAILmaybeapplicable
of tumor necrosis factor (TNF) receptors. Most consist asananti-cancerdrug.However,arecentpaperreported
of a cysteine-rich extracellular domain, a membrane- that TRAIL induces apoptosis in human hepatocytes,
spanning domain, and a cytoplasmic domain containing indicating that substantial liver toxicity might result if
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544 Cell Death (Apoptosis)
TRAILwereusedinhumancancertherapy.Beforeitcan to staurosporine-induced apoptosis. (3) Caspase-2 is re-
beusedinaclinicalsetting,itwouldbenecessarytounder- quiredfortheformationoffemalegermcellsandlossof
standthemolecularmechanismsbywhichTRAILtrans- thecaspase-2generendersBcellsresistanttogranzyme
ducessignalsfromitsreceptorinhumanhepatocytes. B-induced cell death. (4) Patients suffering from au-
toimmune lymphoproliferative syndrome (ALPS) type
II, which display immune regulatory defects, have mis-
III. APOPTOTIC PROTEASES, CASPASES sensemutationsincaspase-10.Caspase-10mutantshave
less caspase activity and block Fas- and TRAILR1-
Intheexecutionphaseofapoptosis,caspaseproteasesare mediatedapoptoticpathways.(5)Bothcaspase-1−/−and
activated by various apoptotic stimuli. Caspases belong caspase-11−/− mice are resistant to endotoxic shock,
tothecysteineproteasefamilyandwereoriginallyiden- such as results from LPS treatment, and their gene
tified as homologues of the ced-3 (cell death abnormal) products are required for the production of cytokines:
gene product (an executor of apoptosis in Caenorhabdi- caspase-1isneededforsecretionofIL-1αandcaspase-11
tis elegans). Indeed, most caspases induce apoptosis if for secretion of IL-1α and β. As discussed above, each
theyareoverexpressedingrowingcellsandcelldeathcan caspaseisimportantforbothvariousdevelopmentaland
beblockedbycaspase-specificinhibitors.Therefore,cas- pathologicalprocesses.Knockoutanalysesforothercas-
pasesareacceptedasexecutorsforapoptosisinmammals pase genes are still under investigation and will reveal
andthispathwayappearstobeconservedbetweenspecies. individual in-vivo functions for each caspase in the near
Caspases are synthesized as zymogens and the active future.
enzymes are generated by proteolytic cleavage of these
caspase precursors. X-ray crystallographic studies have
shown that active caspases are heterotetramers consist- IV. SIGNAL TRANSDUCTION OF DEATH
ing of two large subunits (∼20 kDa) and two smaller FACTOR-MEDIATED APOPTOSIS
(∼10kDa)subunits.Sofar,14caspaseshavebeencloned
from mammalian sources and divided into three sub- Asdescribedearlier,Fas-orTNFRI-mediatedapoptosisis
groups based on primary structure, phylogenetic analy- triggered by binding of the cognate death ligands (Fig. 2).
sis,andsubstratespecificity.Caspasescleaveproteinson In general, apoptosis induced by most death factors can
the carboxyl side of aspartic acid with sequence speci- stilloccurinthepresenceofinhibitorsofproteinorRNA
ficity. For example, caspase-1 shows preferential cleav- synthesis,suggestingthatthedeathfactor-mediatedapop-
age at Asp in the Tyr–Val–Ala–Asp sequence, whereas toticprocessesproceedwithoutdenovosynthesisofeither
caspase-3cleavesatlatterAspintheAsp–Glu–Val–Asp. proteinsorRNAsunliketheprocessesinvolvedingrowth
Caspasesareclassifiedasinitiator,effectorandcytokine- anddifferentiation,andthatallofcomponentsforapop-
releasingcaspases,accordingtotheirfunctions.Initiator toticsignalingareconstitutivelypresentincells.Thereis
caspases include caspase-2, -8, -9, and -10. These have anessentialdomainrequiredfortransductionofthedeath
large prodomains, which interact with specific adapter signals into cells, so-called death domain (DD), found
moleculestoconverttheprecursortotheactiveform.Ef- in the cytoplasmic region of both Fas and TNFRI. Im-
fectorcaspases,includingcaspase-3,-6,-7,and-14,have munoprecipitationanalyseshavesuggestedthataprotein
shortprodomainsandareactivatedbyactiveinitiatorcas- complex,designatedasthedeath-inducingsignalingcom-
pases.Thisregulatorymechanismisknownasaprotease plex(DISC),isrecruitedtothecytoplasmicdomainofFas
cascade.Othercaspasesfoundinmammalsappeartoserve following the interaction of Fas with Fas ligand (Fig. 2).
ascytokine-releasingenzymes. Using the yeast-two hybrid technique with the cytoplas-
Gene disruption experiments have revealed the phys- mic region of Fas or TNFRI as baits, several molecules
iological functions of individual caspases in vivo. (1) that specifically bind to the cytoplasmic region of these
Caspase-3-and-9-deficientmiceshowembryoniclethal receptors have been discovered. FADD (Fas-associating
phenotypes with neuronal hyperplasia, indicating that protein with death domain)/MORT-1 is a small adapter
caspase-3 and -9 play an important role in neu- proteinwithamolecularmassof26kDaandadeathdo-
ronal development. Moreover, their embryonic fibrob- main at the its C-terminus. FADD is recruited to trimer-
lasts are resistant to staurosporine-, etoposide-, UV-, ized cytoplasmic region of Fas and binds to Fas via in-
and dexamethasone-induced apoptosis but not to Fas- teractions between the death domains (Fig. 2). Nuclear
mediated apoptosis. (2) Caspase-8-null mice are embry- magnetic resonance (NMR) studies have shown that the
onic lethal and established cells from these mice are re- death domain of Fas consists of six antiparallel, amphi-
sistant to cell death through death receptors including pathic α-helices arranged in a novel fold. Because there
Fas, DR3, and TNFRI, whereas these cells are sensitive aremanychargedgroupsfromaminoacidsontheprotein
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CellDeath(Apoptosis) 545
FIGURE2 SignaltransductionofFas-andTNFRI-mediatedapoptosis.
surface,thebindingbetweendeathdomainsmaybeme- toTRADDandisinvolvedintheactivationoftranscrip-
diatedbyionicinteractions.Deletionmutantexperiments tionalfactorNF-κBthatisresponsibleforstimulatingthe
haveshownthattheN-terminalregionofFADDbutnot proliferation of thymocytes (Fig. 2). Although RIP has a
its death domain is necessary for transducing death sig- kinasedomainattheNterminus,thiskinasedomainisdis-
nals. In addition, the FADD with deletion of the N ter- pensableforactivatingNF-κB.Severalanalysesindicate
minus works as a dominant-negative mutant against the that NF-κB appears to protect against TNFRI-mediated
Fas-mediated system. Therefore, this region has been apoptosis via up-regulation of c-IAP2 (cellular inhibitor
designated as death effector domain (DED). Similarly, of apoptosis protein 2). Up-regulated c-IAP2 appears to
the DD-possessing protein, TRADD (TNFRI-associated bind TRAF2 (TNF receptor-associated factor 2), which
death domain protein), has been found as an adapter canbindRIPandinhibitapoptoticsignalingfromTNFRI
moleculethatspecificallybindstoTNFRI.UnlikeFADD, (Fig. 2). Both negative and positive apoptotic signals from
TRADDdoesnothaveDED,butisessentialformediating TNFRI might be necessary for fine tuning the decision to
apoptosis induced by TNFRI. Subsequently, it has been dieornot.
shownthatTRADDbindstoFADDviaDDinteractions Twoindependentapproachesinvolvingtheyeasttwo-
(Fig.2).Thatis,TRADDisrecruitedtothecytoplasmicre- hybridexperimentswithDEDofFADDasabaitandpu-
gionoftrimerizedTNFRIthatconsequentlybindsFADD. rificationoffactorsbindingtothecytoplasmicregionof
Thus,FasandTNFRIutilizethesametransducerandshare Fas, have revealed that procaspase-8 binds to the DED.
theapoptoticmachinerydownstreamofFADD.Thesig- Procaspase-8containstwoDEDmotifsattheNterminus
nalthroughTNFRIismorecomplexthanthatofFasbe- through which it binds to FADD and a caspase homol-
causeofthediversityofsignalsfromTNFRI.Forexample, ogous region at the C terminus (Fig. 2). Indeed, DISC
RIP(receptor-interactingprotein),originallyidentifiedas contains both FADD and procaspase-8, and recruited
aFas-bindingproteinwithDDmotif,preferentiallybinds procaspase-8 is processed to the active enzyme near the
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546 Cell Death (Apoptosis)
inner plasma membrane. Several lines of evidences sug- the apoptosome (Fig. 3). Apaf-1 binds to procaspase-9
gest that procaspase-8 may be proteolytically processed via CARD motifs and procaspase-9 is processed using
to the active form by simple oligomerization. However, both Apaf-1 and energy from dATP/ATP hydrolysis to
the precise mechanism is still unclear and may require convert procaspase-9 to the active form. Cytochrome c
unknownadditionalfactor(s)thatmaybeinDISCoron appearstoplayaroleinovercomingtheinhibitionofthe
the inner plasma membrane, may be required to gener- Apaf-1 active site masked by the WD-40 repeat. Once
ate active caspase-8 more efficiently. Once caspase-8 is activated,effectorcaspasesdownstreamofcaspase-9are
activated in cells, it cleaves effector procaspases located sequentially activated (Fig. 3).
downstreamofitsuchascaspase-3,-6,and-7inorderto Ithasrecentlybeenshownthattherearetwocelltypes
amplify the apoptotic signal (Fig. 2). Finally, cells show with different sensitivity to Fas signaling induced by an
a variety of apoptotic features such as described above, agonisticanti-Fasantibody.IntypeIcells,procaspase-8is
duetocleavageofmorethan100substrateproteinswhich rapidlyactivatedfollowingreceptorengagement,whereas
contributestoDNAfragmentation,lossofmitochondrial intypeIIcellstheactivationofprocaspase-8isdelayed,
function, and the maintenance of nuclear structure and althoughbothtypeIandtypeIIcellsshowsimilarkinetics
plasmamembrane. ofFas-mediatedapoptosisandlossofmitochondrialfunc-
An alternative apoptotic pathway via mitochondria tion.Inaddition,Bcl-2inhibitsapoptosisintypeIIbutnot
has been found by the in vitro reconstitution assay (de- type I cells. Why do these cells die in a similar manner
scribed in section V). Based on a dATP/ATP-inducible toeachotherregardlessoftheamountofactivecaspase-8
cell-free system, three factors responsible for process- and yet show different blocking activity by Bcl-2? One
ingprocaspase-3havebeenpurifiedandidentified.These explanationisthattherearetwodistinctpathwaysinFas-
include Apaf-1, which is found as a mammalian homo- mediated apoptosis, a direct pathway from caspase-8 to
logue of CED-4 known to be another executor of apop- effectorcaspaseintypeIcellsandacaspase-8-mediated
tosisinC.elegans,cytochromec(Apaf-2)thatismainly mitochondrial pathway in type II cells. It is likely that
present in the mitochondrial intermembrane space, and Bcl-2 is only able to block the latter pathway. However,
procaspase-9 (Apaf-3). Cytochrome c is released from thishypothesisisstillcontroversialinviewofthereport
the intermembrane space of mitochondria into the cy- thatphysiologicalFasligandbutnotanagonisticantibody
toplasm during apoptosis induced by a variety of apop- killsbothtypesofcellssimilarlyregardlessoftheBcl-2
toticstimuli,includingDNA-damagingagents,proteinki- expressionlevel.Theseresultsmightdependontheeffi-
nase inhibitors (staurosporine), and death receptors (Fig. ciencyofprecisetrimerizationofFas.Theevidencethat
3). Moreover, the release of cytochrome c is blocked therearetwodistinctpathwaysintheFassystemhascome
by anti-apoptotic proteins belonging to the Bcl-2 fam- from the analyses of Bid-deficient mice. If Bid, a Bcl-2
ily such as Bcl-2 and Bcl-xL (Fig. 3). Recent obser- family member, is cleaved by caspase-8, truncated Bid
vations have indicated that cytochrome c-deficient mice (tBid) can translocate from the cytosol to mitochondria,
showanembryoniclethalphenotypewithdefectsinox- subsequently, cytochrome c is released, executing apop-
idative phosphorylation. In addition, their embryonic fi- tosis.Administrationofanagonisticanti-Fasantibodyto
broblasts are resistant to stresses such as UV irradia- wild-type mice in vivo causes death with hepatocellular
tion, serum withdrawal and staurosporine. The mech- apoptosisandhaemorrhagicnecrosisintheliverwithin3
anism by which cytochrome c is released from mito- hours,whereasBid-deficientmicesurvivetreatmentwith
chondria by apoptotic stimuli remains elusive, although thisantibody.Inaddition,hepatocytesfromBid−/− mice
some hypotheses, including opening of a specific chan- areresistanttothisantibodyinvitro,suggestingthatthe
nel for cytochrome c, alteration of the permeability tBid-mediated pathway via mitochondria predominantly
transition pore (PTP) that regulates inner mitochondrial works in hepatocellular apoptosis induced by agonistic
membrane potential, or swelling and subsequent rup- Fasantibody.Itwillbeinterestingtodeterminewhether
ture of the outer mitochondrial membrane, have been ornotApaf-1−/− andcaspase-9−/−conditionalknockout
proposed. mice (because of the embryonic lethal phenotype) show
Apaf-1 possesses a region homologous to the a similar response as Bid-deficient mice to the adminis-
procaspase-prodomain, known as the caspase-recruiting trationoftheanti-Fasantibody.Toinvestigatetheactual
domain (CARD) at the N terminus, a region homolo- signalingpathwaysactivatedbythephysiologicalFaslig-
gous to CED-4 in the middle part and WD-40 repeat andinvivo,itwillbenecessarytoadministersolubleFas
structure that appears to be involved in protein–protein ligandtoBid-deficientmice.
interactions. The released cytochrome c interacts with Some death receptors also activate caspase proteases
two cytosolic proteins, Apaf-1 and procaspase-9, and byligationwiththeircognateligands.LikeinTNFRIsig-
dATP/ATPinthecytoplasmtoformacomplexknownas naling,DDregionsofactivateddeathreceptorsincluding
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CellDeath(Apoptosis) 547
FIGURE3 Apoptoticpathwayviamitochondria.
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548 CellDeath(Apoptosis)
DR3andTRAILR2recruitDISC(thatconsistsofFADD radiation and treatment with ceramide (N-acyl-erythro-
and procaspase-8) via the DD of TRADD. Unlike Fas, sphingosine).Inaddition,extractsfromcellsactivatedby
TNFRI,andDR3,TRAILR1cantransmitapoptoticsig- Fas in the presence of a caspase inhibitor do not cause
nals and activate caspase-8 in FADD-deficient fibroblast DNA fragmentation in the cell-free reaction. Indeed,
cells. This suggests that TRAILR1-mediated apoptosis extracts from proliferating cells, in the presence of
iscaspase-8-dependentandFADD-independent,although recombinant active caspase-3, induce nuclear apoptosis,
an unidentified FADD-like adapter molecules cannot be indicatingthatthefactorsresponsibleforapoptoticDNA
ruledoutatpresent.TheresponsemediatedbyDR6has fragmentation are downstream of the caspase cascade.
not,asyet,beenwellcharacterized. It emerged that these factors form a complex composed
In conclusion, ligand-induced trimerized death re- of heterodimeric proteins of caspase-activated DNase
ceptors activate an apical protease, procaspase-8, via (CAD)/DNA fragmentation factor 40 (DFF40)/caspase-
FADD or an unidentified functional homologue. Acti- activated nuclease (CPAN) and an inhibitor of CAD
vated caspase-8 can sequentially activate effector cas- (ICAD)/DFF45(discussedinthefollowing).
pasesresponsibleforcleavingavarietyofdeathsubstrates Asdescribedabove,ifdATPismixedwithextractsfrom
such as ICAD/DFF45 (discussed below), lamin, fodrin, proliferatingcells,theextractshavetheabilitytoinduce
andpoly(ADP-ribose)polymerase.Inadditiontothedi- nuclear apoptosis mediated by caspase-3 activation in a
rectpathwayfrominitiatorcaspasestoeffectorcaspases cell-freereaction.Usingthissystem,Wang’sgrouphave
throughFas,activecaspase-8cantransducedeathsignals purifiedthefactorsresponsibleforprocessingprocaspase-
intomitochondriathroughthetranslocationofcaspase-8- 3 and identified three gene products, namely Apaf-1,
cleavedBidfromcytosoltomitochondria.UptakeoftBid cytochrome c and procaspase-9. Thus, biochemical ap-
into mitochondria promotes the release of cytochrome c proachesusingcell-freesystemsforapoptosishasdemon-
tothecytosolbyunknownmechanisms.Oncecytochrome stratedseveralimportantaspectsinthefieldofapoptosis.
c isreleased,itcangenerateactivecaspase-9viaforma- During apoptosis, biochemical procedures have fre-
tion of the apoptosome. Thus, death receptors appear to quentlyshownlossofmitochondrialfunction.Inorderto
usetwodifferentpathwaysforparticulartissuesinsome clarifytheregulationsofmitochondrialapoptosis,isolated
situations. The two different pathways ensure the death mitochondriahavebeenused.Duringtheearlystagesof
signalsareamplifiedandtargetcellskilled. studyinthisfield,thefunctionofmitochondriainapop-
toticprocesseswasoverlookedduetonoapparentchanges
onmorphology.However,theobservationsthatBcl-2(an
V. CELL-FREE SYSTEM IN APOPTOSIS anti-apoptotic protein) is abundant in the mitochondria,
and that apoptotic processes are often accompanied by
The establishment of cell-free systems has provided us a decrease in the mitochondrial membrane potential and
with detailed insight into the cognate molecular mech- by mitochondrial swelling led to a more detailed inves-
anisms of various cellular functions, including gen- tigationofmitochondrialfunctionduringapoptosis.Fur-
eral/specific transcriptional regulations, RNA editing, thermore,apoptogenicfactors,suchascytochromecand
protein synthesis and protein degradation, and overall apoptosis-inducingfactor(AIF)thathadbeendiscovered
metabolic pathways. The well-established biochemical asanuclearapoptosis-inducingflavoproteinusingacell-
hallmark of apoptosis is chromosomal DNA degrada- free system, are released from the intermembrane space
tionresultinginmultimersof180bp-nucleosomalunits. ofmitochondriaintothecytosoltoinduceapoptosis.Re-
Neitherprotein-norRNA-synthesisinhibitorsblockFas- cently,anotherfactorsecretedfrommitochondriaduring
mediatedapoptosis,suggestingthatallofthecomponents apoptosis, called Diablo/Smac, has been reported. This
forapoptoticinductionthroughFasarepresentincellsin factor suppresses the functions of proteins belonging to
latentforms. theinhibitor-of-apoptosis(IAP)family,inhibitingthepro-
A cell-free system for apoptosis was first established teaseactivityofcaspasesincludingcaspase-3,-7and-9.
by exposing isolated nuclei to various extracts and Celldeathislikelytobeacceleratedasaresultofinhibition
monitoring nucleosomal DNA fragmentation. That is, ofIAP.Thus,thereappeartobevariousstepstoensurecell
when isolated nuclei from healthy cells were treated killing.Howisthelossofmitochondrialfunctioninduced
with extracts from dying cells (but not from growing bythevariousapoptoticstimuli?Forexample,Bax(Bcl-
cells),theyshowedapoptoticfeatures,includingnuclear 2-associated X protein), which belongs to a member of
morphology with peripheral condensation of chromatin thepro-apoptoticBcl-2family,inducesapoptosis.When
and nucleosomal DNA fragmentation. This cell-free addedindividuallytopurifiedmitochondria-freecytosol,
system can be reproduced using the extracts from cells neithermitochondrianorBaxcanindividuallyinducethe
subjected to different apoptotic stimuli, including UV activation of caspases that lead to nuclear apoptosis. In