Chapter12 Maintenanceof Genomic Integrity andthe Development of
Cancer Whenfirstinterpreting theramifications of DNAand the genetic
code .. . Wetotally missed the possible roleof enzymesinrepair...l
later came torealisethatDNAissopreciousthat probably many distinct
repair mechanisms could exist. Francis H.C.Crick,molecular
biologist,1974 The capacity toblunder slightly isthe real marvel of
DNA. Without
thisspecialattribute,wewouldstillbeanaerobicbacteriaand there would
be no music. Lewis Thomas, biologist, 1979
Thefactthathumantumorformationisacomplex,multi-stepprocess
reflectsthemultiplelinesof
defenseagainstcancerthathavebeenestablished within our cells,each
maintained by the hard-wiring of a complex regulatory circuit. The
human body-actually, its individual cells-must entrust the
maintenanceof
theseanti-cancerdefensestotheirmoststable,reliableconstituents:
DNAmolecules. Over extendedperiods of time,DNAsequences are the
most fixed,unchangeable components of a cell; most of its other
parts are in constant flux,being created and broken down
continuously. Following this logic, it is really the stability of
DNA molecules that underpins the most robust defenses against
cancer. Because there are multiple cellular lines of / 463 Chapter
12: Maintenance of Genomic Integrity andthe Development of Cancer
defensethatdependonDNAstabilityandbecausethebreachingofeach defense
usually requires a rare mutational event, the probability of cell
populations'advancingallthewaytotheneoplasticstatemustbeastronomically
small. So the cancerphobe can rest easy at night, reassured by the
multiplicity of cellular and tissuedefensemechanismsthat evolution
hasassembled toprotectus
fromneoplasia.Butthereisatroublinginconsistencyhere:ifthenumberof
anti-cancer defense mechanisms were truly as great asdepicted in
this text,and if the breaching of each of these defenses were
usually dependent on rare mutational events, then cancers should
never strike human populations. Yetthey do. II Western
populations,in which deaths frominfectiousdiseasesarerelatively
infrequent,about 1 person in5 isdestinedtodiefromoneor another
formof cancer.So,cancer cellpopulations accomplish what
seemstobetheimpossible-acquiringasubstantialarrayofmutant(andmethylated)allelesovera
period of several decades. Researchers working in Seattle,
Washington, attempted to resolve this inconsistency asfarback
as1974. They proposed that the only resolution of this logical
quandary must depend on a drastic increase in mutation rate:cell
populations en route to becoming malignant must carry genomes that
are farmore mutable than the genomes of normal human cells-a
condition sometimes termedthe mutator phenotype. Such speculation
has received increasing support in recent
years,asdifferenttypesofgeneticinstabilityhavebeendocumentedinthe
genomes of cancer cells. Inthischapter,we ,"!illdirectmuch of our
attentiontotwomajor issues. First, how donormalhuman
cellsandtissuesmanagetokeepthemutationrateso
low?Andsecond,howarethestrategiesforsuppressingmutationsthwarted
during human cancer pathogenesis? 12.1Tissuesareorganized
tominimize the progressive accumulation of mutations On a number of
occasions throughout this text, we have described the effects of
carcinogensandtumorpromotersontargetcellsthroughoutthebody.
However,the specificbiologicalidentities of these target cells have
never been spelledout. Asitturnsout,knowledgeof thenature of
thesecellsiscriticalto
understandinghowgenomeintegrityismaintained.Toexplorethisissue,we
needtodelvedeeply intotheorganization of tissuesand
thevarioustypesof cells that formtissues. Their biologicalbehavior
furnishesus \"lithinSights into
thestrategiesexploitedbytissuesandcellstominimizetheaccumulationof
genetic lesions. Asdescribed earlier (Section 11.6), a common
scheme seems to explain the construction andmaintenanceof many
tissuesthroughout thebody. Withineach tissue,arelativelysmallnumber
of cellspopulateitsstemcellcompartment. These self-rene\"ling cells
may constitute a minute fraction ofthe entire cell population
\"Iithina tissue,sometimes asfewas0.1to1%of thetotal.Intruth,in
mosttissues,thesenumbersrepresentnothingmorethanpoorlyinformed
guesses.Becausestemcellsarepresentinverysmallnumbers,haveappearances
that arenot particularly distinctive,and areoftenscattered among
other celltypes \"Iithintissues,they aredifficult toidentify
andstudy. Consequently, much of what isdescribedbelow rests on
inference rather than on direct observation of stem cells and their
properties. We "villbriefly review the discussions of Cha pter
11concerning stem cells. Here, however,wefocus on the normal stem
cells \"Iithintissues rather than the pool 464 Tissuesareorganized
tominimizemutations STEMCELLhighlyFigure 12.1Tissue organization
and COMPARTMENTtransit-amplifyingdifferentiatedcellprotection of
the stemcellgenome cellscellsdeath Theorganizationof manyepithelial
- C) - CJ - G- (-\,,/
..........c.., - .';::! IJ / -"' l----...G-G ,--'.v - -
\}'-''\., ,,/ '- - v- G - C)L' - G_i""".............-..'\..- . _Q
_() ,,/ l- ,_Q_("')o,,,,;on,' 1\ - '-) mitosIs '"'
...............li - 61- (-.'./ '"j_(-\----eo- " .. ,I,,_,1\' '\.,
,,/ '--- ., ----eo- " ("\',--'?'L .........._ - Q - C:: ----eo- c;)
\ ... -L-..J frequent mitosispost-mitotic cells exposed
shieldedfromto toxic agents toxic agents of cancer stem cells that
reside within a tumor. Asis the case in tumors, the stem cells in a
normal tissue are self-renewing, since at least one of the two
daughters of adividing
stemcellwillretainthephenotypeexhibitedbythemothercell prior tocell
division,Inmany tissues,the second daughter celland
itstransitamplifying descendants will pass through a substantial
number of cell divisions
beforeenteringintoapost-mitotic,highlydifferentiatedstate.Theseactively
dividing cells, which serve as intermediates between a stem cell
and its differentiateddescendants,maythereby
generatedozens,possibly evenhundreds,of differentiated descendants
of the second daughter cell(Figure 12,1). The exponential increase
in the number of transit-amplifying cells means that a stem cell
needs to divide only on rare occasion in order tomaintain a large
pool of end-stage,highly differentiated
cellsinatissue.Therefore,whileone might think that stem
cellsparticipate incontinual cycles of growthand division,the
realityisusuallymuchdifferent:thetransit-amplifyingcellscreatethegreat
bulkof mitoticactivityinmanytissues.SincetheDNAreplicationoccurring
during each cell cycle isinherently prone tomaking errors, this
scheme reduces the risk that mutations will accumulate inthe
genomes of the stem cells within a tissue. Inmany
epithelialtissues,thedifferentiated epithelialcellsareespecially
vulnerable to damage, since they form cell sheets that line the
walls of various ducts
andcavitiescontainingtoxicmaterial.Inthecasesof thecolonandthebile
duct,theepithelialcellsconfrontfecalcontentsandhighlycorrosivebile,
respectively. The cells lining the alveoli in the lungs cope every
day with
particulatesandpollutantsintheair.Thekeratinocytesinourskinareexposed
directlytotheoutsideworldandhenceareliabletosustainseveraltypesof
damage,including that inflicted by ultraviolet radiation. The
differentiated end-stage cells (see Figure 12.1)in these and other
tissues have a finite lifetime and are discarded sooner or later.
Some cell types may simply age tissuesseemstoconformtothescheme
shownhere.Asindicated,eachstemcell (blue)dividesonly
occasionallyinan asymmetricfashiontogeneratea new stemcelldaughter
anda transit-amplifyingdaughter.Thesestemcellsare
oftenshieldedanatomically fromtoxic
agentsThetransit-amplifyingcells (green)undergorepeatedroundsof
growthanddivision,expanding exponentially.Eventually,theproductsof
thesedivisionsundergo further
differentiationintopost-mitotic,highly
differentiatedcells(red).Thehighly
differentiatedcells,whichareoftenin directcontactw
ithvarioustoxicagents, areshedw ithsomefrequency;hence, anymutant
allelesthat ariseinthese cellswillbelost sooneror later,from
thetissue.Thismeansthatthegenomes of
stemcellsareprotectedthroughtwo mechanisms :
stemcellsrarelydivide,and theyareprotectedanatomically from
noxious,potentiallymutagenic influences. 465 Chapter 12:Maintenance
of Genomic Integrity andthe Development of Cancer and losetheir
viability,being worn out fromcarrying on the active business of
thetissue.Forexample,redbloodcellshaveanaveragelifetimeofapproximately
120 days,after which they are scavenged by the spleen and broken
down and their contents recycled or excreted. The epithelial cells
in the colon live for 5 to7 days before they are induced toenter
apoptosis and are sloughed off into the lumen of the intestine. The
keratinocytes in our skin die within 20 to 30 days of being
formed,and they are shed continually in small flakes of dead skin
(see, forexample, Figure 2.6A). Hence,the transit-amplifying cells
may wellrun an increased riskof sustaining mutations becauseof
their highmitoticactivity,and their
differentiatedprogenymayoftenbelocatedinmutagenicenvironments.However,anygenetic
damage that the transit-amplifying cellsandtheir
differentiatedprogeny have sustained willhave little consequence
forthe tissue as a whole: sooner or later, these cellsareflushedout
of the tissue,and once they die,any mutations they may have
accumulated disappear with them. The dynamics of stem cells and
their progeny are illustrated most graphically by the stem cells
and the enteroeytes (the differentiated epithelial cells)ofthe
small intestine and the colon. These cells and their behavior have
been described earlier in thecontext of our discussionsof the Ape
tumor suppressor gene and ~
catenin(Section7.11).Here,wereturntothemonceagaintoillustrateother
principles.Recallthatthestemcellsareembeddeddeepwithinthecrypts
(Figure12.2A).Theretheyarewellout of harm'sway,being shielded
fromthe mutagenic contents of the intestinal lumen by a thick layer
of mucus secreted by cells in the crypt. This mucus, which is
formed from highly glycosylated proteins
termedmucins,createsajelly-likebarrierthatpreventsthecontentsof the
intestinallumenfrompenetratingdeepintothecrypt(e.g.,seeFigure12.2B)
and illustrates yet another strategy by which mutations can be
minimized in the genomesof stem cells:evolution has created
mechanisms by which stem cells
areanatomicallyshieldedfromtheactionsoftoxins,includingcarcinogens.
(Thus, mice that have been genetically deprived of the gene
encoding Muc2, the mostabundant gastrointestinalmucin,areprone
todevelopadenomas inthe small intestine, many of which progress to
adenocarcinomas.) This confers further protection on the genomesof
stem cells,complementing themechanism mentioned earlier, in which
the descendants of these cells, which may have sustained mutations,
are driven out of the crypts and eliminated after a period of 5 to
7 days (Figure12.2C). In theory, the stem cellcompartment within a
tissue has an inexhaustible
abilitytogeneratedifferentiatedprogenywithouteversufferingdepletion.However,almost
inevitably,a stem cellVlrillbe lostfromthiscompartment through one
or another mishap. This gap in the ranks must be filledby other
stem cells. More specifically, both daughters of a surviving stem
cell will need toretain the phenotypeof their
mother,whichthereforeundergoesasymmetrical division (Figure12.3).
Thismay alsohaveimplications forgenome maintenance, aswe will
seebelow. 12.2Stem cellsarethe likely targetsof the mutagenesis
that leadsto cancer Thepropertiesof
theintestinalcells(seeFigure12.2)alsoprovideimportant clues about
the identities of the cells that are the likely targets of
carcinogenesis.
Havingexcludedthemoredifferentiatedcells(becausetheyarerapidlydiscarded),
we must shift our focustothe long-livedcells and cell lineages
within epithelial tissues. Actually,we already have come across
evidence that points to the centralroleof
long-livedcellsintheprocessof carcinogenesis.InSection 1l.13, we
read about experimental protocols used to induce skin cancer in
mice. 466 Stem cellsaretargetsof cancer pathogenesis (A)lumen of(B)
smallintestine Izone of differentiation zone of PCOI; T";OC 150
proliferat i ve cells Tc =12h 4-6 actual 5stem cells cellposition
anethcells villus - 4 circumference (C) 96hours
Onesuchprotocolinvolvedpainting apatchof skinwithaninitiatingagent,
allowing the patch toremain untouched forsome months, and then
painting it repeatedly with TPA,a potent skin tumor promoter. Cells
that had been exposed
totheinitiatingcarcinogen"remembered"thatexposureoneyearlaterby
undergoing proliferationandformingaskinpapillomainthepresenceof the
promoter. In the skin, as in many other epithelial tissues,the
long-lived cells are those in the stem cell compartment.
Provocatively,thenumberof skinpapillomasandcarcinomasinducedbythe
mouseskincarcinogenesisprotocol(seeFigure11.28)isnotred ucedifthe
mouse skin istreated with 5-fluorouracil (5-FU)shortly after being
exposed toa mutagenic initiating agent. Since 5-FU selectively
kills actively cycling cells,this indicates that the cell targeted
by carcinogenic mutagens during initiation is not t tips of villi
bottom of crypts ! 40minutes24hours48 hours Figure 12.2
Stemcellsand the organization of gastrointestinal crypts
(A)Theschemeofti ssue organizationdepictedinFigure12 .1 is
illustratednicel ybytheorganizati onof
theepithelialcells-enterocytes-inthe smallintestine(seealsoFi
gure115). The 4to6stemcellslocatednearthebottom
ofthecrypts(red)areshieldedfromthe
contentsofthesmallintestinebytheir locat ionandbymucus
thatpreventsthe entrance of fluids fromtheintestinal lumenintothe
crypt (seepanel8).The stemcellsspawnalargenumber (-150)
ofhighlyproliferativetransit-amplifying
cells(yellow,green),whichdivideevery 12hoursor
so.Theirdivisionyields approximately 3500enterocytes(blue), w
hichcoverthe villus-the f ingerli ke
structurethatprojectsintothesmall intestine. Theenterocytesare
continuously migratingtowardthetipof
thevillus,wheretheyundergoapoptosis andareshedintothelumenof
thesmall intestine.Thenumbers totherightof the cryptindicate
thecellpositionnumber fromthebottom of the crypt.(B)Copious amounts
of protectivemucus(dark purple) aresecretedbythecellslining
comparablepitsinthewallof the stomach;similarmucus,composedof
highlyglycosylatedproteinstermed mucins,isfoundinthecryptsof
thesmall andlargeintest ine.(C)Theemigrationof transit-amplifying
cellsfromthecryptsof thesmallintestine canbetrackedby injectinga
doseof 3H-thymidine (ie., radioactivethymidinecarryinga tritium
atom)into amouseandfollowingthe
resultingincorporationofradiolabelinto
DNAbyautoradiography;radioactive decayisindicatedbydark silver
grains. (Incorporationoccursonly during abrief periodof time. )
Seenherearethe cellsin thecryptsof theduodenumof the mouseat
theindicatedtimesafter injectionofthe3H-thymidineCellsthat
multipliedonlya smallnumber of times afterinitialincorporationof
3H-thymidine remainheavilylabeled(broad arrows), w hilethe
greatmajority underw ent multiple additionaldi visionsafterlabeling
(duringthechaseperiod)andtherefore
exhibitdilutedradiolabeling.After four days,virtually allof
thecellgenOmesthat weresynthesizedatthebeginningof the
experimenthavebeencarriedout of the cryptsto thetipsof thevilli .
(A,courtesy of C.S.Potten;B,fromB. Youngand LWHeathetaI.,W
heater'sFunctional Hi stology,4thed.Edinburgh:Churchill Li
vingstone,2003;C,fromC.S.Potten, Phi!.Trans.Royal Soc.LondonB
353821 - 830,1998) 467 ---------- ----------Chapter 12:Maintenance
of Genomic Integrity andthe Development of Cancer (A)stemcell I\
transit-stemcell- amplifying cell 1st2nd daughterdaughter (C) I
/\growth of organ, increase instemcell number, /\symmetric
divisions l /\/\ /\ maintenance/\ of organ size, constant number of
stemcells,/\ asymmetric divisions L (8) II I symmetric -1
division:/\ /\/\/\ - - - - T---- -I /\/\/\/\ asymmetric division
conversionof /-transit- amplifying cellto stem cell Figure 12.3
Asymmetric and symmetric divisions of stemcells (A)
Ingeneral,duringnormalti ssue f uncti on,itappearsthata stem cell
willusually divideasymmetrically, withoneof its daughters
remaininga stem cell(blue) w hiletheother(green)proceeds to spawn a
fl ock of transi t-ampl ifyingcell s (not shown;seeFi gure121)(B)
Inthe eventthat severalstemcell s ina ti ssuearelost,some
ofthesurvivingstem cell s may dividesymmetri call y
inordertore-populatethestemcellpool withthe propernumberofcell
s.Asseenhere,threestemcellshavebeenlost (redcrosses,toprow)from a
poolof sevenstemcells.Thesubsequent symmetric divisions
undertakenby thesurviving stemcellsmake possi ble a regeneration of
theoriginalpopulati on sizeofthestemcellpool. Alternati vel y,the
lossofa stemcell(red cross,third row) may cause its
transit-amplifyingsistertorevertbackto a stemcell(bottom). (e)
Similarly,whenanorganisgrowing,thenumberof stemcell s must
increaseproporti onately,requiringsomestemcellstoundergo.
symmetricdivi sions. intheactivecellcycleatthetimeof
initiationandshortly thereafter, lending weight tothe notion that
thetarget forinitiation isa celltypethat divides only occasionally.
Analysesof several types of leukemia suggest that the
initialtargets of
carcinogenesisinthehematopoieticsystemarealsostemcells.Themostdramatic
example is provided by chronic myelogenous leukemia (CML) . As
described earlier,the Philadelphia(PhI)chromosome, whichresults
fromareciprocal
chromosomaltranslocationthatfusesthebcrandablgenes(seeSection4.6)
,is observedinalmostallcasesof
thisdisease.Extensiveevidencepointstothis particular translocation
as the genetic lesion that initiates this disease. A number of
distinct hematopoietic celltypes within a CMLpatient may carry the
PhIchromosome. Included are lymphoid cells (both B and T
lymphocytes), aswellascellsof the myeloidlineage(including
neutrophils, granulocytes,the megakaryocyte precursors of
platelets, and erythrocytes). This observation provides persuasive
evidence that the cell type in which the translocation originally
occurredwasthecommonprogenitorof allof
thesehematopoieticcelllineages-the pluripotent stem cell that
servesasthe precursor formany typesof
hematopoieticcells(Figure12.4).Likeavarietyofotherstemcells,this
hematopoieticstemcell(HSC)isthoughttohaveaverylonglifetimeinthe
hematopoietic system, more specifically inthe bone marrow.
Intheparticular case of CML, a stem cell that has suffered a
criticalmutation-formation of the PhIchromosome-retains the option
todispatch its progeny into a number of
distincthematopoieticcelllineages.Yetotherindicationsof theroleof
stem cells as targets for tumor formation come from other types of
hematopoietic disorders(Sidebar 12.1). 468 progenitor r---------- -
-:THYMUS I @I ' Stem cellsaretargetsof cancer pathogenesis
Highlycompelling observationsof stem cells'role in cancer
derivefromtransgenicmicein which the expressionof
anactivatedrasoncogeneislimited to either thekeratinocytestem
cellsin the skin(which in thiscasearelocated in hair follicles)or
the keratinocytes that have begun to enter into a terminally
differentiated state. When the transgene directs expression of
therasoncogene in thestem
cells,themicedevelopmalignantcarcinomas.Incontrast,whenthe same
oncogene is expressed in the differentiating keratinocytes, benign
papillomas are formed,and these tend toregress. NKcell _
________________ , n common lymphoid dendritic cellc @t- @ multi
potentmulti potent hematopoietichematopoietic : . .
stemcellprogenitor ..""'4: monocyteosteoclast neutrophil n
@eosinophil@ common myeloid @ basophil progenitor o L-. @{ @ f [
p'''''''' megakaryocyte eryth rocyte ....." ...... . ;........''.'J
I STEMCELLCOMMITTEDPROGENITORSDIFFERENTIATEDCELLS Figure12.4
Hematopoietic differentiation Ourcurrent
understandingofhematopoietic celldifferentiationteachesa number
oflessons.(1)Itindicatesthata singlecelltype-the
multipotenthematopoieticstemcell(HSC;left)-is capableof
generatingvirtuallyallofthecelltypesinthebloodandinthe
immunesystem.(2)Itshowsthata singlestemcelltypecanspawn
mUltipletypesof"committed"stemprogenitor cells,inthiscase, thetwo
stemcelltypesthatarecommittedtogeneratinglymphoid
andmyeloidcelltypes.(3)Itshowsthat self-renewalability(curved
arrows)isnotconfinedtoa singlestemcelltypeina tissue;instead,
incertaintissuessuchasthisone,"committedprogenitors"
(ie.,thelymphoidandmyeloidstemcellsshownhere)aswellas
someoftheirdescendantshaveself-renewalcapability.Thefact thata
patientsufferingfromCML(chronicmyelogenousleukemia)
oftenexhibitsseveraldistinctdifferentiatedlymphoidandmyeloid
celltypescarryingthePh 1chromosome(anda BCR-ABL
translocation)providesstrongindicationthatthisabnormal
chromosomewasinitiall y formedinsomemultipotentHSCor
progenitor.(FromB.AlbertsetaI.,MolecularBiology oftheCell, 4th ed .
NewYork:GarlandScience,2002 .) 469 Chapter 12: Maintenance of
Genomic Integrity andthe Development of Cancer Sidebar 12.1Blocked
differentiation is a frequent theme
inthedevelopmentofhematopoieticmalignancies Therearedozensof
examplesof malignanciesinanimals andinhumans whereinhibitionof
differentiationfavors the appearance of neoplasias. Possibly the
first of these
situationstobedefinedgeneticallyinvolvedtheavianerythroblastosis
virus, a retrovirus that encodes two
oncoproteins:itserbBoncogenespecifiesaconstitutivelyactive .version
of the epidermal gro'ATthfactor(EGF)receptor (see Section 5.4),
which drives the proliferation of er)Tthroblasts
(precursorsofredbloodcells);whileitserbAoncogene encodes a nuclear
receptor (ahomolog of the thyroid
hormonereceptor),whichinhibitsdifferentiationofthe
hyperproliferating erythroblasts created by erbB. Similarly,
inhumanacutemyelogenousleukemia(AML).,alarge variety of genetic
lesionsfoundinthe leukemic cells have
beenassignedtotwofunctionalclasses:thosethatare required to drive
the proliferation of the myeloid precursor cells, and others that
are required in the same cells to block subsequent differentiation.
Inthemegakaryoblasticleukemias(amalignancyof platelet precursor
cells)encountered with some frequency inDown syndrome patients, the
gene encoding the GATAI
transcriptionfactorhasbeenfoundtobefrequently mutated,preventing
the proper maturation and differentiation of theseprecursorsof
platelets. These fewexamples point to the notion that the exit of
cells fromstem cell compartments must be impededinorder
fortumorigenesis to succeed.
Notaddressedbytheseobseniationsaretheprecise
identitiesofthestemcelltargetsoftransformation.In
manycases,thetargetisnotlikelytobethepluripotent hematopoietic stem
cell,but instead one of itsderivatives that isalready committed
toone or another lineage of differentiation. Such "committed
progenitors" (see Figure 5.4) normallymay
haveSignificant(butlimited)self-renewal
capacityandarenotyetfullydifferentiated,andthereby
canbeconsideredstemcells.Theirtransformationfrom normal to tumor
stem cells involves, among other changes, anacquisition of
unlimited self- renewal capability. Thesevarious strandsof
evidence,obtained fromseveraltypesof tissue, converge on the
conclusion that self-renewing cells of various types are the
targets of the genetic changes thatlead,sooner or later,to the
formation of tumors. In someinstances,thetargetcellsmay bestem
cellswith Wllimitedself-renewal capacity;inothers, committed
progenitors, which normally have only a limited ability to renew
themselves, may acquire unlimited self-renewal capability
duringthecourseof tumorigenesis.Thisidea,inturn,mayexplainsomeof
the complex epidemiology of certain types of human cancers, such as
breast cancer (Sidebar 19 0). 12.3Apoptosis, drug pumps, and DNA
replication mechanisms offer tissuesa way tominimize the
accumulation of mutant stem cells The apparently prominent role
played by normal stem cells as targets fortransformation indicates
that the genomes of these cells must be protected by
whateverbiologicalandbiochemicalstrategiesthesecellsandthetissuesaround
themcanmuster. Wehavealreadycome
acrosstwosuchstrategies:therelatively infrequent replication of
stem cell DNAand the placement of stem cells in anatomically
protected sites. Still,these mechanisms do not seem to suffice, so
the organism has developed yet other strategies. The stem cellsin
themouseintestinal crypts(seeFigure12.2)and mammary
glandsrepresentespeciallyattractiveobjectsforstudyoftheseprotective
strategies.Inthe caseof the crypts,the need foradditional
protective mechanisms is clear: the enterocyte stem cell lineages
in the crypts ofthe mouse small
intestinehavebeenestimatedtopassthroughasuccessionof
1000growthand-division cycles during a lifetime, and each ofthese
cycles exposes the stem cells to various types of genetic damage.
Similarly,in the human gut, the numberof celldivisionsoccurringeach
yeargreatlyexceedsthetotalnumberof cellsresiding at any time within
the entire body;this enormous mitotic activity,most of which
involves tranSit-amplifying cells,must also depend on many
successivestemcelldivisions,althoughinthehuman casetheapproximate
number isnot known. 470 Stem cellsminimize risk of mutation
Oneprotectivemechanismissuggestedbytheresponsesof stem cellsinthe
cryptstomassivegeneticdamage. In theintestinalcrypts of
themouse,stem cellsthathavesuffered genetic damageinfli
ctedbyX-rayswillrapidlyinitiate apoptosis rather than halt their
proliferation and attempt to repair the damage.
Themotivehereseemstobeassociatedwiththeerror-pronenatureof DNA
repair.Aswewilllearn
later,theDNArepairapparatusishighlyefficientbut hardly perfect,and
therefore often leaves a residue of unrepaired or incorrectly
repaired lesionsinthechromosomal DNA.If such lesionsareencountered
by the DNAreplication machinery,they may causemutant DNAsequences
tobe
copiedandpassedontodaughtercells,includingthosethatwillthemselves
become stem cells. So,
ratherthanriskthisoutcome,stemcellsinthemouse crypts are primed to
activate apoptosis in response to DNA damage. It is unclear whether
stem cells inother tissues are similarly poised toenter apoptosis.
Yetanother mechanism issuggested by a commonly used technique for
separating stemcellsfromthebulkof
cellsinatissueviafluorescence-activated
cellsorting(FACS;seeFigure11.13).Stemcellsefficientlypumpoutcertain
fluorescencedye molecules, whilethesecells'differentiated
derivativesdoso muchlessactively.Asaconsequence,afterexposureof
cellpopulationsto suchdyes,thestem
cellsfluorescemuchmoreweaklythanallother cellsin thesepopulations.
The active excretionof these fluorescentdyemolecules isdue tothe
actions of a plasma membrane protein termed Mdr1(multi-drug
resistancei), which was firstdiscoveredbecause itisexploited bymany
cancer cellstopump out,and therefore acquire
resistanceto,chemotherapeutic drug molecules. Theunusually high
levels of Mdr1expressed by many types of stem cells seem
torepresent
astrategythattheyusetoprotecttheirgenomesfrompotentiallymutagenic
compounds that may have entered into their cytoplasms from outside.
The mechanism of asymmetric DNA strand allocation may alsoplay an
importantroleinpreventingthestemcellsincertaintissuesfromaccumulating
geneticdamage.Theexperimentalobservationssupportingthisproposed
mechanismarestillfragmentary.Nonetheless,itispresented
here,becauseof its interest and potential importance
tounderstanding cancer pathogenesis.
Therationalebehindthisstrategy,firstproposedin1975,derivesfromthe
moleculardetailsof theDNAreplicationoccurringinstem cells.
Wehavejust revisited the model that when stem cells divide, the
division is usually asymmetric,in that one daughter cellremains a
stem celland the other enters into a
differentiationpathwaybyproducingtranSit-amplifyingcells(seeFigure12.1).
Ideally,thegenomethatisdonatedtothedaughterthatremainsastemcell
shouldbeaffordedmoreprotectionthanthegenomethatispassedontothe
daughter destined for differentiation, because descendants of the
latter are destinedtobe discardedsooner or
later.AsillustratedinFigure12.5A,theasymmetric allocation of DNA
strands can help toaccomplish this aim. The idea here is based,
once again, on the fact that DNA replication is inherently
error-prone.Bysomeestimates,eachtimeacellpassesthroughS phaseand
replicatesitsDNA,severalnucleotidesubstitutionsoccurpercellgenome
becauseDNApolymerasesmakemistakesthatescapesubsequentdetection and
repair.(Intruth, this number may greatly underestimate the errors
in DNA replication.)Consequently,DNAstrandsthat werenot synthesized
during the most recent cycle of DNA replication-the "conserved"
strands-are more likely
toretainwild-typesequencesthanarethose"nonconserved"strandsthatare
indeed the products of this DNA synthesis. This suggests that in a
well-designed tissue, the DNA strands that have not been created
byrecent DNAreplication should beretained bythe daughter cellthat
remainsinthestemcellcompartment,whilethoseDNAstrandsthatarethe 471
Chapter 12:Maintenance of Genomic Integrity andthe Development of
Cancer (A)conservednonconserveCi(B) strandst:.:.and r""""=--__""
,tem,," -WI\ 3H-thymidineBrdU3H-thymidine
labeledlabeled+BrdUlabeledmoo stem cells/Itransit- I\(D)
//amplifying+CD(])"'"00
etc.etc.etc.etc. (C)conserved strand ;; (E) symmetric division
I\L 2nddaughter isretained c;- In stem cellcompartment r,andbecomes
astemcell asymmetr/'ctransit-divisionamplifyingt L(])cell JIone of
the recentlyreplaced DNA strands becomes (F) t!aconserved strand
that is ,,;retainedinthe .stem cellcompartmentCD (DI\I\I\
etcetc.etc.
productsofDNAreplicationshouldbeallocatedtothedaughtercellwhose
descendantsaredestinedtodifferentiateandeventuallydie.AsFigure12.5A
makes clear,one DNA strand(the conserved, "immortal" strand)can, in
principle,betransmittedindefinitelythroughalineageof stemcellsby
suchasymmetric segregationof
DNAmolecules.Stateddifferently,stemcellsmaycarry DNAstrands that
haverepeatedly servedastemplates forDNAreplicationbut areonly
infrequently synthesizedasproductsof replication(atleast sincethe
472 Stem cellsminimize risk of mutations Figure12.5ConservedDNA
strandsandthe stemcellgenome
(A)The"immortalstrand"modeldepictsoneconservedDNAstrand(yellow)ofthechromosomalDNAof
a stemcell(light blue)thatisdonatedtoitsdaughter
cellthatwillremaina stemcellandis
thereforeretainedinthestemcellcompartment;thisconservedDNAstrandisnot
the product
ofrecentDNAreplication.Conversely,the"nonconservedstrand"(red)thatis
indeedtheproduct
ofrecentDNAreplicationwillbeallocatedpreferentiallytothedaughter
cellthatspawnstransit-amplifyingcells(light
green)andthereforeexitsthestemcell compartment;thenew
roundofDNAreplicationaddsa new red strandtothe non
conservedparentalred
strand.ThismodelpredictsthatoneDNAstrandcanpersist indefinitely
withinthestemcellcompartment withoutundergoingreplication.(B)This
predictionisfulfilledinthemousemammary gland.Micecanbeexposedtoa
briefpulseof 3H-thymidineata
timeduringpubertywhentheglandisstillgrowingandthenumber of mammary
epithelialstemcellsiscontinuouslyincreasing,necessitatingsymmetricaldivisions
inwhichbothdaughters of a stemcellbecomestemcells(seeFigure12
.3C)andtherefore,
hypothetically,bothstrandsofDNAareretainedasconservedDNAstrands.Hence,label
incorporatedduringthisperiodmay beretainedindefinitely
inconservedDNAstrands. DNA thathasincorporatedthe
3H-thymidinetradiolabelisdetectedby incubatingti ssueslices w itha
photographicemulsion(darkgrains)-the procedureof autoradiographyFi
veweeks afterinitialexposureto
3H-thymidine,radiolabelisretainedinonly about 2%of the mammary
epithelialcells(left).Atthat time,micecanbeexposedto a pul seof
bromodeoxyuridine (BrdU),athymidineanalogwhoseincorporationinto DNA
canbe detectedby a specificantibody thatrecognizes
BrdU-containingDNA(red staining nucleus, middle).Followingthispul
se,BrdUcanbedetectedinthemajority ofthecellsthatretained
radiolabelfromtheexposureto 3H-thymidine5 weeksearlier (right
panel).Hence,these label-retainingcellsareactivelyproliferating5
weekslater yettheyretaina conservedstrand that wassynthesized5
weeksearl ierandisnotlostfromthesecellsbytherepeatedrounds of
growthanddivisionthat theyareundergoing.(C)Whena
stemcellislost(topright)in
anadult(inwhichthesizeofthestemcellpoolshouldbeconstant),a
survivingstemcell willdividesymmetrically,sothatbothofitsdaughters
willremainasstemcells,thereby reconstitutingthepopulationof
stemcellsinthepool(seeFigure12,3B)Inthisdaughter (right),a
DNAstrandthat waspreviouslynonconservedandthustheproductofrecent
replication(red)willberetainedinthestemcellcompartment
andbecomeanimmortal, conservedstrand(yellow),Hence,thekillingof
stemcellsinanadult shouldmakeitpossible tolabelstemcellDNAinsucha
fashionthat thislabelisretainedindefinitelyinthestemcell
compartment.(D)Thispredictionisfulfilledbythebehaviorofcellsintheduodenumofthe
mouse.Inthecrypts,theenterocytesarenormallyreplenishedbythecontinual
multiplicationofthestemcellslocatednearthebottomof
thecrypts(seeFigure12.2A)As
inpanel(B),theDNAmoleculesinproliferatingcellscanberadiolabeledbya
briefexposure to
3H-thymidineanddetectedsubsequentlybyautoradiography.Normally,allofthe
radiolabeledDNAthatisinitially synthesizedinthecryptsmovesout
ofthecryptstogether withthedifferentiatingtransit-amplifyingcell s
andtheirenterocyteprogeny andhenceis lostfromthecryptsafter
severaldays(seeFigure12.2C).However,ifthe duodenumis
exposedto8grays (Gy)of X-irradiation(whichkillssomeof the
stemcells)before radiolabelling, cellsInthe cryptscanbefound to
retainlabeleven8daysafter a briefpul se ofradioactivethymidine.
Fourexamples of theselabel-retaining cells(LRCs),whicharefound
preciselyinthelocationof stemcellsinthe
crypts,areshownhere(arrows).(E) Inthi s mammary duct,themammary
epithelialcells(M ECs)arestainedfor cytokeratinexpression
(red).AnLRCthatincorporatedBrdUnineweeksearlierisimmunostainedingreen.(F)LRCs
canbefoundinthe"bulge"regionof thehair folliclesof themouse,w
herekeratinocyte stemcellsareknownto reside.Thesecells,w
hichwerebriefly inducedto expressa stable formof greenfluorescen
tprotein(G FP,green)atfourweeksof age,continuetoexpressGFP four
weekslater.Theepithelialcellsarelabeledhereinred(B,fromG.H.Smith,
Development132681-687,2005;D,courtesyof
C.S.Potten;E,courtesyofB.Welmand MA Goodell;F, courtesyof
1Tumbar,VGreco,andE.Fuchs)
adulttissuewasfirstformed),WhileFigure12.5Aillustratesthebehaviorof
a short stretch of DNA, we can imagine that the entire chromosomal
DNA of stem cells behaves in this fashion as well. This model of
asymmetric strand allocation (also called the "conserved-strand"
model)can be tested experimentally. In fact,we have already seen
one manifestation of this behavior in the experiment illustrated
inFigure 12.2C. In that case, 473 Chapter 12: Maintenance of
Genomic Integrity and theDevelopment of Cancer stem cells were
allowedtoincorporate 3H-thymidine fora brief period of time.
TheradioactiveprecursorbecameincorporatedintotheDNAstrandsbeing
replicatedduringthisshortperiodof
time,afterwhichfurtherincorporation ceased.(Such an experimental
protocol is often termed "pulse-chase" labeling.) The fate of the
radiolabeled DNA molecules was then followed through the
techniqueof autoradiography,inwhichaphotographicemulsionisplacedona
sliceof tissue;thisemulsion yieldsareadilyvisualizeddarkgrain
whenever a radioactive atom such as a tritium atom decays. If the
allocation of DNA strands were symmetrical,then we would expect
some of theradiolabel wouldremain behind in the stem cell
compartment and some would be distributed to the differentiating
cells that had left the stem cell compartment..
AsFigure12.2Cdemonstrated,virtuallyalloftheradiolabeledDNAstrands
migrated out of the stem cell compartment together with the
transit-amplifying cellsthat
hadbeguntodifferentiate.Thissupportsthenotionthatthenewly
synthesized strands(Le.,those that were synthesized during the
3H-thymidine pulse)werepreferentially
donatedtothedaughtercellsthatspawnedtransitamplifying cells and
their more differentiated descendants that migrated out of the
crypts. Conversely, we discover that it isextremely difficult to
label the DNA strands that are retained in the stem cell
compartment within the crypts. Actually,there isan alternative
interpretation to this observation: the radiolabel
leavesthestemcellcompartmentbecauseitisrapidlydilutedbyrepeated
cycles of growth and division in the stem cell compartment. This
notion can be tested critically by exposing stem cells to
3H-thymidine at a time when the stem
cellcompartmentisexpanding;undertheseconditions,stemcellsmust
undergosymmetricdivisioninordertoincreasetheirnumber(seeFigure
12.3C),and both radiolabeled DNA strands should therefore be
retained in the stem cell compartment. This is just what isseen
when the mammary epithelial
stemcellsofthemouseareallowedtoincorporate3H-thymidineduring
puberty,whenthemammaryglandisgrowingrapidly(Figure12.SB).Under
theseconditions,theradiolabelisretainedmany weekslaterin thestem
cell compartment, even though these "label-retaining"
cells(LRCs)can be shown to beactivelydividingatthislater
time.(Theradiolabeledstrand inherited from
theirancestorsmanycellgenerationsearlierretainsitsradiolabelinspiteof
repeated intervening cycles of cell growth and division.) Another
test of the conserved-strand model comes fromexperiments in which
some of the stem cells are killed by exposure to X-rays. The
remaining cells in the stem cell compartment willattempt toreplace
the lost stem cells through symmetriccelldivisionsin which
bothdaughtersremain asstemcells(seeFigure 12.3B). Consequently, a
newly made DNA strand, which would normally be allocated to the
differentiating daughter cell, will now be converted into a
conserved strand and retained in the stem cell compartment (Figure
12.SC). If we expose the stem cell compartment of mouse intestinal
crypts to 3H-thymidine during the period of time when the loststem
cellsarebeing replaced,we can indeed label DNA molecules that
subsequently remain within the stem cell compartment foran
indefinite period of time; that is,the labeled molecules are not
"chased" out when the stem cells are exposed subsequently to
non-radiolabeled thymidine precursors (Figure 12.SD). Hence, the
only time in an adult animal that we can introduce long-lived
radiolabel into the stem cell compartment
seemstobewhenweperturbthiscompartmentbykillingsomeofitscells. Under
these conditions, both the immortal DNA strand and the recently
synthesized,non-immortal strand are retained in cells that become
stably ensconced in the crypts of the small intestine.
Label-retaining cells arealso found in other epithelial
tissues(Figure12.SEand F).In spite of this and other evidence, most
of whichhasbeen gathered in the mouse, the "immortal-strand" model
remains largely a matter of speculation for 474 DNA replication
leadst ooccasional copying errors Sidebar12.2 Some
carcinogensmaying cells,as would normally be its fate
producemutantcellsthroughtheir(see Figu're12.3BJ.However,this
sister ability to be cytotoxic The "immortal
cellmayhappentocarryamutant
strand"modelmakespredictionssequenceduetoaDNAreplication
abouthowcertaincarcinogenswork.errorthatoccurredduringthemost
Wecanimagine,forexample,thatrecent S phase.Oncethissister cellis
somecarcinogens,ratherthanbeingrecruitedintothestemcellcompartdirectlymutagenic,actthroughtheirment,theDNAstrandcarryingthis
abilitytobecytotoxicand' thuskillmutant sequence may then be chosen
cells within a tissue, including some ofto become an "immortal" DNA
strand, itsstem cells.Inthe event that such athereby ensuring
tl1atthismutation is
carcinogenkillsastemcellrecentlynowpermanentlyestablishedinthe
formedbymitosis,thesisterofthatstem cell compartment. stem
cell,formed by the same mitosis,Wehaveencounterednongenomay
beretained inthe stem cell com toxiccarcinogenspreviouslyinthe
partmentratherthanbeingdis contextofthediscussionoftumor
patchedintothepoolofdifferentiat- promoters(Section11.14).There,we
mosttissuesandrequiresfarmoreexperimentalvalidationbeforewecan
acceptitasawell-establishedfact.Theimmortal-strand theory,if
further
validated,alsoholdsimportantimplicationsfortheprocessofcarcinogenesis
(Sidebar 12.2). 12.4Cell genomesarethreatened by errorsmadeduring
DNA replication The design of stem cell compartments and the
behavior of individual stem cells
illustrateseveralbiologicalstrategiesusedbytissuestoreducetheburdenof
accumulated somatic mutations. Thesemechanisms serve toprotect stem
cell genomes,whichconstitute,ineffect,the"germlines"of
tissues.Importantly, these strategies represent only the first line
of defense against genomic damage. The next line of defense isa
biochemical one that depends on the ability of various proteins
torecognize and repair damaged DNA molecules within cells.
Infact,DNAmoleculesareunderconstantattackbyavarietyof agentsand
processes. For the sake of simplicity, we can place these mutagenic
processes in three categories. First, as mentioned above, the
replication of DNA sequences by DNApolymerasesduringtheSphaseof
thecellcycleissubjecttoalowbut
nonethelesssignificantleveloferror.Includedamongtheseerrorsarethose
generatedwhenchemicallyalterednucleotideprecursorsareinadvertently
incorporated intoDNAin placeof their normal
counterparts.Second,even in the absence of attack by mutagenic
agents,thenucleotides within DNAmoleculesundergochemical changes
spontaneously;thesechanges often alterthe base sequence and thus
the information content of the DNA.Finally,DNA molecules may be
attacked by various mutagenic agents, including those molecules
generated endogenously bynormal cellmetabolism as wellas agents of
exogenous origin-chemical species and physical mutagens (X-rays and
UV rays)that
areintroducedintothebodyfromoutside.Wewillreturntothelattertwo
processes in the next sections. The molecular machinery that
isresponsible for replicating almost all chromosomal DNA sequences
has a remarkably low rate of error. The basic replication machinery
in the cellnucleusispoweredby the actions of three polymerases,
pol-a,pol-8,andpol-.(Inall,15distinctDNApolymerasegeneshavebeen
arguedthatsometumorpromoters, such as ethanol, act through their
abilitytocause thedeath of cellsina target tissue, resulting in a
compensatory proliferationbythesurvivingcellsin the
tissue.Now,asweview these promoters in the context of stem cell
biology,wecanspeculatethatmany tumorpromotersmayactthrough their
ability tokillstem cells. Aneven moredangerousagentwouldbea
"complete"carcinogen(Section11.17) -one that is able toact both
asan initiatorthroughitsmutagenicactions
011stemcellgenomesandasapromoter throughitscytotoxiceffectson stem
cells. 475 Chapter 12:Maintenance of Genomic Integrity andthe
Development of Cancer Figure12.6 ProofreadingbyDNA
polymerasesAnumberofDNA polymeraseshavea proofreadingability
thatallowsthemtominimize thenumber ofbasesthataremisincorporatedand
retainedintherecentlysynthesized strand. Thus,asa DNApolymerase
extendsa nascentstrand(darkblue)ina 5' -to-3'
direction(movingrightward),it w illusethe existing3'-OHof
thenascent strandastheprimerforfurther elongation(light
blue)However,ifa base hasbeenmisincorporated(third
drawing),theDNApolymerase,whichis continuouslylookingbackwardto
check whetherithasincorporatedthecorrect basesinthe
growingDNAstrand,can degradeina 3'-to-5' (leftward)direction
therecentlyelongatedstrand(fourth drawing) andundertakeonceagainto
synthesizethisstretchof nascentstrand (bottomdrawing).
catalogedinthehumangenome,andmorearelikelytobefound;aswillbe
apparentlater,many of thesearenotinvolvedinDNAreplicationpersebut
rather in the repair of damaged DNAmolecules.)
Acellhastwomajorstrategiesfordetectingandremovingthemiscopied
nucleotides arising during DNA replication. The first strategy lies
in the hands of
theDNApolymerasesthemselves,whicharestructurallycomplexaggregates
assembledfroma number of distinct protein subunits. vVhilethey
areadvancing down single-strand DNA templates and extending nascent
DNA strands in a 5' -to-3' direction,DNApolymerases such as pol-8
continuously look backward, "over their shoulder," scanning the
stretch of DNA that they have just ized;such monitoring isoften
called proofreading. Should a polymerase detect a copying error, it
will use its 3' -to-5' exonuclease activity to move backward and
digest the DNA segment that it has just synthesized and then copy
this segment once again, with the hope fora better outcome the
second time (Figure12.6). DNApolymerase 5'))' 3'5' direction
template strand of polymerization _ ! elongation of newly
synthesizedstrand 5')) , 3'5' ! misincorporated nucleotide 5' 3'5'
! 3' ..."""""'....- ... .........---5' polymerase movesbackward and
degrades recently synthesizedstrand ! )), 3' .........--...
polymerase moves forward againandundertakes once again to
synthesize proper sequence 476 DNA replication leadstooccasional
copyingerrors +1+ 100...... m >.;; ~50 ~ o 00 24681012 age
(months) Theimportanceof thisproofreadingmechanismfortheprevention
of cancer has been illustrated dramatically by the creation of a
mouse strain whose germline pol-8 -encoding genehas been subtly
altered(by a single aminoacid substitution) . Theresultingmutant
pol-8retainsitspolymerizingactivitybut has lostits3'-to-5'
exonucleaseactivity;thislosseliminates itsproofreading
function.Inacohort of 49micecarrying themutant pol-8
alleleinahomozygous configuration,23developed tumors byone yearof
agewhilenotumors developed inagroupconsistingof
twiceasmanyheterozygousmice(Figure12.7).
Thisexperimentprovidesadramaticdemonstrationthatthemaintenanceof
wild-type genomic sequences,inthiscasebya DNApolymerase,
representsa criticaldefenseagainst the onset of
cancer.Moreover,forus,thisobservation representsthefirstof many
indicationsthatthemutationsleadingtocancer may arise through
endogenous processes rather than being triggered exclusively by
invading foreign carcinogenic agents. Follmvingcloseontheheelsof
theDNApolymerasesandtheirproofreading activities are a complex set
of mismatch repair (MMR)enzymes. These enzymes monitor recently
synthesized DNA in order to detect miscopied DNA sequences that
havebeen overlookedbythe proofreading mechanisms of the
DNApolymerases. The actions of the mismatch repair system become
especially critical in regions of the DNA that carry repeated
sequences. These sequence blocksinclude simple mononucleotide
repeats(such as AAAAAAA),dinucleotide repeats(such as
AGAGAGAG),andrepeatsofgreatersequencecomplexity.Becauseof strand
slippage, which occurs when the parental and nascent strands slip
out of proper
alignment,DNApolymerasesappeartooccaSionally"stutter"whilecopying
these repeats,resulting in incorporation of higher or lower copy
numbers of the repeat sequence into the newly formed daughter
strands (Figure 12.8). Thus, the sequence AAAAAAA,that is, A7,might
well cause a polymerase to synthesize aT6 or Tssequence
inthecomplementary strand. Theresulting
insertionsordeletionsmayeludedetectionbytheproofreading
componentsof theDNApolymerases andaretherefore prime
targetsforrecognitionand repair bythemismatch repair machinery.
Forhistorical reasons, highly repeated sequences inthe genome,often
carrying100ormorenucleotidesperrepeatunit,havebeencalled"satellite"
sequences.Becausethesimple,shortersequencesdiscussedherearealso
found inmany places in the genome, they have been named
microsatellites. A Figure12.7 Proofreadingby DNA polymerase and
cancerincidence Apoint mutationhasbeenintroduced
intothegerm-linecopyofthemouse geneencodingDNApolymerase8,the
mammalianDNApolymerasethatis responsibleforthebulkofleadingand
laggingstrandsynthesis.Thi s mutation,
termed0400A,alterstheaminoacid sequenceintheproofreadingdomainof
thepolymerasebyspecifyingthe replacementof anasparticacidbyan
alanineatresidueposition400of the polymerasemolecule.Shownhereisthe
fateof53wild-typemice(+1+),97 heterozygotes (+I0400A),and49
homozygousmutants(0400AI0400A). Deathsofthemutant homozygotes were
allduetomalignanci es;theseincluded
lymphomas,squamouscellcarcinomas of theskin,andseveralother typesof
cancerthatoccurredrelatively infrequently.Two of theheterozygotes
diedfromcausesthat wereunrelatedto cancer, whilethehomozygous
wild-type miceallsurvivedto the ageof oneyear.
Theirsurvivalcurvesareshownherein thisKaplan-Meierplot.(From
R.E.Goldsby,NA Lawrence,L.E . Hays etal.,Nat.Med.7638-639,2001) 477
Chapter 12:Maintenance of Genomic Integrity andtheDevelopment of
Cancer defective mismatch repair system that fails to detect and
remove stuttering mistakes made by DNA polymerases when copying a
microsatellite will result in the
expansionorshrinkageofitssequencesinprogenycells.Thiscreatesthe
genetic condition known as microsatellite instability
(MIN;Figure12.9), which may ultimately involvechanges inthousands
of microsatellite sequences scattered throughout a cell genome.
(A)(B)TaqMutS + 783TBuige T 5' ___ T T T T T T T::;:_:3' 3' ......
AAAAAAA5' 5' ___ TTTTTT::;::3' 3' ...... AAA A AAA5' TC 5' ___
TCTCTCTC_3' (D)3' ...... domain I (B) 5' ___ T C T C T C 3' ......
AGAGAG5' AG (C) -error r innew ly 1BINDINGOFMISMATCH made
strandPROOFREADINGPROTEINS IDNA SCANNINGDETECTS MutSMutLNICKINNEW
DNA STRAND 1STRANDREMOVAL domainIV 1 (B)REPAIRDNA SYNTHESIS
Figure12.8 DNApolymeraseerrors andmismatchrepair
(A)TheDNApolymerases,notablypol-5,occasionally"stutter, " or
skipabasewithinarepeatingsequenceofDNA (e.g.,a microsateliite
sequence)presentinthetemplate strand(blue) indicatedhere. As
aconsequence,thenewly synthesizedstrand (green) may
acquireanextrabasethatincreasesthelengthof the repeatingsequence
ormaylackabase(toptwo images).Identical dynamicsmay
causesimilarchangesinmicrosateliitesequences wheretherepeatunitisa
TCdinucleotidesegment(bottomtwo
images),oramorecomplexrepeatingsequence(not shown)
(B)Mismatchrepair(MMR)proteins functiontorecognizeand
repairthemistakesmadebyDNApolymerases,including
misincorporatedbasesandinaccuratereplicationofmicrosatellite
sequences.Onepowerfultechniqueto visualizethefunctionsof indi
vidualMMRproteinsusesatomic-forcemicroscopy.Here,the
MutSMMRproteinof thebacteriumThermusaquaticus,ahomolog of anumber
of themammalianMMRproteins,hasbeenvisualized bindingto aDNA
fragmentinto w hichamismatchhasbeen introducedat
aspecificnucleotide site.MutSkinkstheDNAdouble helixasitscansfor
andultimately findsregionsofmismatch, where
itbindsinastablefashion,seenhereasa white pyramid.(C)In
eukaryoticcells,two components of theMMR apparatus,MutSand
MutL,collaboratetoremovemismatchedDNA.Asillustratedin
panelB,MutS(green) scanstheDNAformismatches.MutL then scanstheDNA
for single-strandnicks,whichidentify thestrand
(red)thathasrecentlybeensynthesized;theunder-methylationof
therecentlysynthesizedstrandmayalsoaidinthisidentification. MutL
thentriggersdegradationof thisstrandbackthroughthe
detectedmismatch,allowingfor repairDNAsynthesi s to follow and
generate aproperlymatchedDNA strand.Itisunclear w hether MutL
alsousesother cluesto determinetherecentl ysynthesized DNA
strand.(D) Thefuncti onof theThermusaquaticusMutSMMR
proteinisrevealedinevenmoredetailbyX-raycrystallography.Part
ofthestructure theTaquaticusMutShomodimeric proteinin complex
withamismatchedhelix (red)isshownhere.DomainsI andIVof subunit
Aareindark blue andorange,whilethe correspondingdomains of subunitB
areinlight blue and yellowAn arrow (yellow)indicates
wherephenylalanineresidue39of subunitI
isassociatedwithanunpairedthymidineinoneof the two strands.
Defectsinthehumanhomologof thisproteinplayacriticalrolein
triggeringhereditarynon-pol yposiscoloncancer(HNPCC ),discussed
inSection12.9. (B,fromH. Wang,Y.Yang,M.J. Schofieldet ai. ,
Proc.Nat/.Acad.Sci.USA10014822-14827,2003;C,from B.Albertset
ai.,Molecular BiologyoftheCell,4th ed.NewYork:
GarlandScience,2002;D,fromG.Obmolova,CBan,PHsiehand WYang,Nature
407 :703-710,2000) 478 Cell biochemistry generates mutagens Yet
other, more subtle copying mistakes made by a DNA polymerase, such
as the incorporation of an inappropriate base in a nonrepeating
sequence, may also be detected and erased by mismatch repair
proteins, which arehighly sensitive to
bulgesandloopsinthedoublehelixcausedbyinappropriately incorporated
nucleotides.Themismatchrepairmachinerymustbeabletodistinguishthe
recentlysynthesizedDNAstrandfromthecomplementary "parental"strand
that served as the template;this enables the MMRapparatus todirect
its
attentiontoremovingandthenrepairingtherecentlysynthesizedandtherefore
defectiveDNA strand (seeFigure12.8C) . Mismatch repair involvesthe
excision of the nucleotides that have created the mismatch and a
new attempt at synthesis of this strand.
Workingtogether,thesevariouserror-correctingmechanismsyieldextremely
low rates of miscopied bases that survive to become mutant DNA
sequences. To begin, DNA polymerases make copying mistakes in only
about lout of 105
polymerizednucleotides.The3'5'proofreadingbythepolymerasesoverlooks
aboutlout ofevery102 nucleotidesinitiallymiscopiedbythepolymerase,
thereby reducing the error rate toabout 1 in 107 nucleotides. After
the DNA
polymerasehaspassedthroughastretchofDNA,themismatchrepairproteins
check the recently synthesized DNA strand a second time. The
mismatch repair enzymes fail to correct only about 1 miscopied base
out of 100 that have escaped the attentions of the proofreading
carried out by the DNA polymerase. Together, this yields a
stunningly low mutation rate of only about 1 nucleotide per 109
that havebeen synthesized during DNAreplication. Aswe
willsee,defectsin these
error-correctingmechanismscanleadtobothfamilialandsporadkhuman
cancers. Finally,DNAreplication
holdsyetotherdangersforthegenome.Somemeasurements indicate that as
many 10double-strand (ds)DNA breaks occur per cell genome each time
a cellpasses through S phase. These breaks appear tooccur
nearreplicationforks,ostensiblybecausethesingle-strandDNAatthe
unwoundbut not-yet-replicated portion of the parental
DNAissusceptibleto inadvertent breakage (Figure12.10).Cells have
well-developed mechanisms for
dealingwithsuchdsDNAbreaks,aswewillseelater.Failuretorepairsuch
breaksproperlycan leadtodisastrousconsequences,includingchromosomal
breaks and translocations. 12.5Cell genomesareunder constant attack
from endogenous biochemical processes Most accounts of the origins
of contemporary cancer research contain a strong emphasis on the
actions of carcinogenic agents that enter the body through various
routes, attack DNA molecules within cells, and create mutant cell
genomes thatoccaSionallycausetheformationof
cancercells.Unrecognizedbythese models of cancer pathogenesis are
the mutagens and mutagenic mechanisms of origin.In recent
decades,however, analytical techniques of greatly
improvedsensitivityhaveallowedresearcherstodetectalteredbasesand
nucleotides in the DNA of normal cells that have not been exposed
to exogenous mutagens. The resultsof these analyseshave caused
aprofound shift inthinking abouttheoriginsof most mutant
genespresent inthe genomesof human celis,because they have shown
that endogenous biochemical processes usually make fargreater
contributionstogenomemutation than doexogenousmutagens.
Sincemutagenic events,independent of their origin,arepotentially
carcinogenic, this has forceda rethinking of how many human cancers
arise.
ThestructureoftheDNAdoublehelix,withitsbasesfacinginward,offersa
measure of protection fromalltypesof chemical attack by shielding
itspotentially reactive chemical groups, notably the amine side
chains of the bases, from BAT25 t colon1\1\ 1\_ colon...,.JV vv
tumor tumor - larger size Figure 12.9Detectionof microsatellite
instability Microsatellite instability (MIN)oftencausesan
expansionor contractionof the sizeof a microsatelliterepeatsequence
. Inthe analysisshownhere,thesizeof a mononucleotiderepeatis
revealedusing a PCR(polymerase chainreaction),in
whichtheprimersbindto sequences flankingtherepeatonbothsides.The
BAT25sequence,whichislocatedon humanChromosome4q 12,consistsof
thesequenceTITIxTxTITIxT7xxT25, where"x"indicatesa nucleotideother
thanlBecauseof errorsmadebythe polymeraseusedinthePCRreaction,the
productsof a reactionshow a Gaussian
distributionoflengthsgroupedarounda
PCRproductthatistheactuallengthof thegenomicDNAsegmentbeing
amplifiedThi s analysisshowsthe lengthsof a microsatelliterepeatina
womansufferingfromHNPCC (hereditarynon-polyposis coloncancer),
whohasbeendiagnosedwithboth colonandbreastcarcinomas;theDNA
ofnormaltissueadjacenttothecolon carcinomagraphhasalsobeenanal
yzed. Thisanalysisrevealsa clearincrease in
sizeofthemicrosatelliterepeatinthe coloncarcinoma(/eft'vvard
shift), w hile thebreasttumor exhibits a microsatellite
repeatthatisprecisel y thesameas normal,controlDNA.(Thisobservation
strongl y suggeststhat thebreast carcinoma,unlike the
coloncarcinoma, isunlikel y to have beencausedby MIN.)
(FromA.Muller, lB. Edmonston, DA Corao et aI. ,Cancer Res.
621014-1019,2002 ) 479 Chapter 12: Maintenance of Genomic Integrity
andtheDevelopment of Cancer Figure 12.10 Double-strandDNA breaks at
replicationforks During DNArepli cation,theDNAmoleculesare
especiallyvulnerabletobreakageinthe single-strandedportions of
themolecule nearthereplicationforkthat have not
yetundergonereplication. Theresulting breakisfunctionallyequi
valent toa double-strandbreakoccurringin an
already-formeddoubleheli x, inthat the breakleaves twohelices
unconnectedby eitherstrand . direction of movement of 4-
replication fork
- - newly synthesizedstrands !single-strandbreak various
mutagenic agents. In spite of this clever design, DNA molecules are
subject tochemical alteration and physical damage.Some of this
damageappears tooccur through the actions of hydrogen and
hydroxylions that arepresent at lowconcentration(-10-7
M)atneutralpH.Oftencitedinthiscontextisthe processof
depurination,inwhichthechemicalbondlinkingapurinebase (adenineor
guanine)todeoxyribosebreaks spontaneously (Figure12.11A).By some
estimates,asmany as10,000purine basesare lost bydepurination each
day in amammalian cell.(Thisamountstomorethan1017 chemically
altered nucleotides generated each day in the human
body!)Depyrimidination occurs ata 20- to100-foldlowerrate,but
stillresultsin asmany as500cytosineand thymine bases lost per
cellper day.Estimates of the steady-state levelof basefree
nucleotides present in a single human genome range from 4000to
50,000. At the same time, deamination may occur,in which the amine
groups that
protrudefromguanine,adenine,andcytosineringsofthebasesarelost.This
dearninationleadsrespectivelytoxanthine,hypoxanthine,anduracil(Figure
12.11B). The uracil, forinstance, may subsequently be read asa
thymine during subsequent DNA replication, thereby causing a C-T
point mutation, known as a transition mutation, in which one
pyrimidine replaces another. The bases generated by deamination are
allforeignto normal DNA,and consequently can be
recognizedassuchandremovedbyDNArepairenzymes.However,any such
altered bases that escape detection and removalby these repair
enzymes represent potential sources of point mutations. Spontaneous
deamination of 5-methylcytosine-the methylatedformof cytosinethat
weencounteredearlier(Section7.8)-occursevenmorefrequently, yielding
thymine (see Figure 12.11B) . This creates a serious problem for
the DNA repair apparatus, since thymine (unlike the other three
products of deamination
describedabove)isacomponentofnormalDNA,andthe T:Gbasepairmay
thereforeescape detection,survive,and ultimately serveastemplate
duringa subsequent cycle of DNAreplication, leading toa C-to-T
point mutation. In fact,this deamination of 5-methylcytosine
represents a major source of point mutations inhuman DNA.Byone
estimate,63% of the point mutations inthe genomesof tumorsof
internalorgans(i.e. ,in thosetissuesshielded fromUV
radiation)arisein CpG sequences. Among mutant p53 alleles, about
30%seem to arise from CpG sequences present in the wild-type p53
allele.[Tobe accurate, this percentage isinflated somewhat bythe
factthat during lung carcinogenesis,methylated CpG sequences are
also favoredtargets forattack bychemically
activatedformsofbenzo[a]pyrene(seeSection12.6),apolycyclicaromatic
hydrocarbon (PAH) present in tobacco smoke. Hence, not allmutations
arising at CpG sites derive fromdeamination events.] 480 Cell
biochemistry generatesmutagens (A)GUANINE o H20 t oP- O- CH\.NI21I
L: ,/'H0 - . 0HNN H- II \0dRdR dR mispairing of
8-oxo-dGdeoxyguanosine (dG)8-oxo-deoxyguanosine with deoxyadenosine
(dA) (8-oxo-dG) NH2oCH3 spontaneous33 deaminationH'
b--.-..CHNIoxidation
r
-
OH CHN.-,;.OH . -_j'.H OAHH oNOH
OHII dRdRdR deoxy S-methyl-cytosinedeoxythymidine glycol
(dS'me)(dTg) Figure12.12 Oxidation of basesin the DNA Theoxidati on
offormedbytheoxidationofdG, canmispair withdeoxyadenosine DNAbases,
whichoftenresults fromtheacti onsofreactive oxygen(dA)rather
thanforming a normalbasepairwithdeoxycytosine species(ROS),can
bemut agenic intheabsence of
subsequentDNA(dC).Hence,if8-oxo-dGisnotremovedfrom a doubleheli
x,the repairreacti ons.(A) Two frequentoxidationreactions invol
veDNAreplicationmachinery may inappropriately incorporate a dA
deoxyguanosine(dG),whichisoxidi zedto 8-oxo-deoxyguanosinerather
thana dC oppositeit.resultingina C- Apoint mutation. (8-oxo-dG);
anddeoxy-5-methyicytosine(d-5' -mC), thenucleotide" dR"signifies
deoxyribose inallcases.The purines areshownin thatis
presentinmethylatedCpGsequences.Uponoxidation,thevarious shades of
red andbrown, whilethe pyrimidines areshown latterinitially forms
anunstablebasethatrapidly deaminates,invarious shades of green .
yieldingdeoxythymidineglycol (dTg).(B) The8-oxo-dG,whichis 12.6Cell
genomesareunder occasional attack from exogenousmutagensand their
metabolites Aswehaveseenrepeatedlyin thistext,cellular
genomesarealsodamagedby exogenous carcinogens, including various
types of radiation as well as molecules Sidebar 12.4 Oxidation
products in urine provide an
estimateoftherateofongoingdamagetothecellular genome Byrecent
estimates,the genomes of some human cellssufferasmany as103
oxidativehitsaday,about10foldlessthantherateatwhichdepurinationofbases
occurs.ThereSUltingoxidizedbasesarelargelybutnot totally removed
and replaced\.v1ththe appropriate normal bases. Rats cells suffer
about lO-fold more oxidative hits per cell per day in their genomes
than do human cells because
theyhaveabouta7-foldgreatermetabolicrate(Figure
12.13).Anyunrepairedoxidativelesions\.viIIaccumulate
\.vithtime,especiallyinthegenomesof cellsthatarenot mitotically
active. 8-oxo-dGisthemostfrequentlyobservednucleotide product of
oxidative damage. It seems that 1 to 2%of these oxidized
nucleotides failto be removed by the DNArepair apparatus. Oxidants
may oxidizethe nucleotide precursor
ofdGpriortoitsincorporationintoDNA;theoxidized
nucleosidetriphosphatemaythenbeincorporated instead of dGTP into
the DNA. Alternatively, oxidants may attack the guanine baseafter
itsincorporation into DNA.
TheimportanceoftheoxidizeddGTP(Le.,8-oxo-dG
triphosphate)isindicatedbythefactthataspecial enzyme-MTH1-is used
bymammalian cells todegrade this oxidized DNAprecursor;mice lacking
MTH1develop tumorsata3to4timeshigherratethantheir wild-type
counterparts.(Yetanotherhighlyspecializedenzyme, called
MUTYH,excises adenines that have been misincorpo rated opposite
8-oxo-dG bases in the DNA.)The 8-oxodG excised fromDNA islargely
excreted in the urine. Unfortunatel
y,theanalysesofoxidationproductsof DNAhave been subject to anumber
of artifacts,including notably the inadvertent oxidation of DNA
andnucleosides in vitro.On one occasion, aliquots of one DNA
preparation were sent to21laboratoriesin Europe formeasurement of
8-oxo-dG content; the resulting analyses yielded estimates ranging
over a factor of more than 200. The estimates of the numbers of
oxidized bases incell genomes have fallen
dramaticallyinrecentyears.Nonetheless,thenewer,more
conservativeestimatesplacethesteady-statelevelof8oxo-dGresiduesintheDNAisolatedfromanaverage
humancellatabout3000.Thesesteadycstatelevelsare comparable to the
level of chemically altered bases that are formedintheDNAof
targettissuesof laboratory animals that have been exposed to high,
carcinogenic doses of compounds such as aflatoxin and heterocyclic
amines. 48 Chapter 12: Maintenance of Genomic Integrity andthe
Development of Cancer 8 0 u >, 0> Q) 6c 'E>; >,Os; 4
f-' 6..E0 f-'c : : : J ~ 0 2C V'>>tJ0:::;g;g I fibers
constructed of microtubule proteins. The fibers together form a
metaphase spindle. The metaphase spindle, in turn, isa bipolar
structure in which each half
spindleisconstitutedofmicrotubulefibers ,manyof whichextendfromthe
kinetochoresonthechromosomes(thenucleoproteinbodiesassociatedwith
the centromeric DNA of the chromosome) back to the centrosomes; the
latter are
responsiblefororganizingtheentiremetaphasespindlestructure.Whenthis
apparatusisworkingproperly,thespindlefiberspullsisterchromatidpairs
apart,sothat each chromatid movestowardoneof thetwocentrosomes.
This ensuresthat thetwodaughtercellsthat willeventually
ariseaftercelldivision receive precisely equal allotments of
chromosomes (see Figure 8.3B). Thiscomplexprocessof chromosome
segregationismonitoredbyaseriesof checkpoint controls, which ensure
initially that precisely h.vocentrosomes and t\.vohalf
spindlesform;that each chromatidinapair associates withitsown,
distinct half spindle;and that chromatid separationisnot
allowedtoproceed unless and until allpairs of chromatids
areproperly aligned on the metaphase
plate.Whenthesecheckpointmechanismsfailtoimposequalitycontrolon
chromosomal segregation, both sister chromatids in a pair may be
pulled to one ortheothercentrosome(theprocessof
nondisjunction).Asaconsequence,
oneofthesubsequentlyarisingdaughtercellsmaybecomehaploidforthis
chromosome and the other triploid. Alternatively, a chromatid may
fail to attach to a spindle fiber and may simply be lost from the
genomes of descendant cells. More widespread karyotypic chaos may
occur if the spindles themselves are not properly assembled.
Aberrant mitoses, which result from inappropriate spindle
organization,were noticed asearly as1890and,in
retrospect,represented the first clue that cancer cells are
genetically abnormal. In normal interphase celis, asingle
centrosome canbe visualized in the cytoplasm (Figure12.37A);during
fromchromosomalarms.(A)Inthe colorectalcarcinomasstudiedhere,
analysesofa largenumberoftumors haverevealedthatmanytumorshave
lostheterozygosity (LOH; seeSection 7.4)ata substantialnumberof
chromosomalloci . Ontheabscissa, 0.3allelicloss,forexample,refersto
tumorsinwhich30%ofthelocithat werepreviouslyheterozygous,as
revealedbyanalysesofchromosomal markers,nolongerexhibit
heterozygosity(red bars).Mostofthis LOHisattributable to
thelossofwhole chromosomes. Incontrast.amongthe
tumorsafflictedwithmicrosatellite instability(MIN;blue
bar),thelossof allelesandhencethelossofentire
chromosomesisnegligible.(B)In colorectaltumorcellslinesthatexhibit
CIN,asgaugedbythelossof chromosomalmarkers(seepanelA),the
rateofinactivationoftheHPRT (hypoxa
nthinephosphoribosyltransferase)geneis virtuallyzero(firstfour
bars,red).Incontrast,inthosethat exhibitMIN,therateofmutationofthis
geneissignificantandisoccasionally 100-foldhigherthaninCINtumorcell
lines(lastfour bars,blue)(A,from C.Lengauer,K.W.Kinzlerand B.
Vogelstein,Nature 396:643-649, 1998, andB. Vogelstein,E.R.Fearon,
S.E.Kernet ai,Science244207-211, 1989;B,fromC.Lengauer,K.W.Kinzler
andB.Vogelstein,Nature396643-649, 1998,andJ.R.Eshleman,E.Z.Lang,
G.K.Bowerfindetal.,Oncogene 1033-37,1995) 12.10 Widespread
chromosomal aberrations are not present in all types of human
cancer cells Thekaryotypes of carcinoma cells and of hematopoietic
tumor cells showastrikingdiscrepancy:theepithelialcancercells
almostinvariablyexhibitwidespreadkaryotypicchaos,
includingavarietyof nonreciprocaltranslocations,dele.tions of
chromosomal arms; and duplications of others. In
contrast,thekaryotypesof hematopoietictumor cellsare often
diploid,withtheexception of one or tworeciprocal translocations
that seem tobe responsible for initiating the
cancerortriggeringaspecificstepof tumorprogression (e.g.,the one
creatingtheBCR-ABLoncogene). Therefore, . chaotic karyotypes arenot
required for the formation of all types of human malignaricies. It
is highly likelythatthe smallnumber of observable karyotypic
alterations found in most hematopoietic cancer ' cellsdonot,on
their own,sufficetoenable fullneoplastic
proliferation.(Inonecase-thatof chronicmyelogenous
leukemia-theacquisitionoftheBCR-ABLoncogeneis often followedduring
blast crisis relapse by the loss of p53 function;point mutations
cause thisloss,and they are,of
course,karyotypicallyinvisible.)Moreover,hematopoietic
tumorcellshavenotbeenreportedtosufferfrom micro satellite
instability. It therefore remains unclear what genetic mechanisms
enable hematopoietic cells toacquire theentireensembleof mutant
allelesneededin order for them to proliferate asfullyneoplastic
cells.Indeed,wedo knowwhethertheformationofhematopoietic tumors
requires as many genetic changes as those needed for the formation
of solid tumors (see Section 11; 12). 513 Chapter 12:Maintenance of
Genomic Integrity andtheDevelopment of Cancer Figure12.37
Centrosomesand the organization of the mitotic spindle
Centrosomesareresponsiblefor organizingthemicrotubule
spindlefibersatmitosis. (A)Inimmortalizedbut nonmalignant
interphase cells,thepresenceof a singlecentrosomecanbe
detectedinthe cytoplasmthroughuseofanantibody
thatdetectspericentrin,a
centrosome-associatedprotein(red).Thiscentrosomeisnormally
duplicatedattheGl/Stransitiontogeneratethetwo centrosomes found
atthepolesof themitoticspindle.(B)Incontrast,duringinterphaseof
humanbreastcancercells,multiplecentrosomescanoftenbeobserved.
Thesewilloftencreatemultipolar spindles(seeFigure1238) whensuch
cellsenter mitosis.(C) Thepair of centriolesthatformsthe coreofeach
centrosomecanbebestseenusingtransmissionelectronicmicroscopy
(TEM),inthiscaseof a humancoloncarcinomacell.Fourcentriolesare
seenhereincrosssection(small arrows),whil e a sideview of a fifth
(largearrow)isapparent,indicatinga deregulationofcentriolenumber.
Thenuclearmembraneisseenabove.(AandB,fromGAPi han,
A.Purohit,l.Wallaceetai,Cancer Res.58:3974-3985,1998;
C,courtesyofM.l.DifilippantonioandTRied.) mitosis,twocentrosomes
arearrayedatoppositepoles within thecell.Cancer cells,however,often
show marked defects in this organization, ineluding multiple
centrosomes at interphase (Figure 12.37B and C).The result may be
mitotic
spindlesthathavemultiplepolesratherthanthetwoseeninnormalcells
(Figure12.38AandB)andthedivisionof thenormalchromosomalarrayof
chromosomes among three or more daughter cells(Figure12.38C). Asa
further consequence, the resulting mis-segregation of chromosomes
into daughter cells may lead to wildfluctuations in chromosome
number and overall karyotype.In one survey of a series of
87different rumors,81of these showed abnormalities in centrosome
number or in the microstructure of individual centrosomes; such
defects were never encountered in normal cells usedascontrols in
this study. It seems that once the complex apparatus designed to
ensure proper chromosomal segregation has been damaged, such damage
is irreversible. For example, as was seen in Figure 12.35,the
enormous cell-to-cell variability inthe number of Chromosome 8
copies in certain breast cancer cells indicated that chromosome
instability(CIN)persistedinthesecellslongaftertumorprogressionhad
reachedcompletion.Inthisrespect,CINdiffersfromthebreakage-fusionbridge(BFB)cyclesdescribedearlier(Section10.4),which
seem toplague the genomes of cancer cells fora limited window of
time during tumor progression and then cease once cells succeed in
acquiring telomerase and thereby stabilize their karyotypes.
Inrecent years,some of themolecular defectsthat contribute
tovarioustypes of chromosomal instability have come to light.Not
surprisingly, the duplication of centrosomes isclosely coordinated
with cell cyele advance; it seems to occur at or near the Gj/S
transition. More specifically,an increasing body of evidence
indicates that centrosome duplication iscoordinated insome way by
cyclinEandA-containingcyelin-dependentkinase(CDI0.5n,4.0n, 0 ).
Whena double-strand DNAbreakissustainedinchromosomal DNA, H2AX
molecules (but not the other histones) become
phosphorylated,primarilybytheATMand AIRkinases,ona
specificserineresiduelocatedfouraminoacidresidues
fromthecarboxy-terminalendofthesemolecules;such phosphorylated
H2AXisobserved ina largechromosomal region(involving as much as 2
megabases of DNA)flanking
thebreak(Figure12.41).(Thesetwokinasesarealso
responsibleforphosphorylatingandtherebymobilizing
p53;Figure9.13.)TheresultingphosphorylatedH2AX (sometimestermedy ~
H 2 A X )servestoattractDNArepair proteins,such asBRCAIand
NBS1,aswellasat least four othersthataidinthetaskof
rejoiningtheDNAends(see alsoFigure 12.30). Mice that have lost the
H2AX gene (because of inactivationof
thisgeneinthemousegermline)areviablebut stunted in growth. Their
cells are unable to execute
homology-directedrepair(Section12.10)and areprone toaccumulate
structurally abnormal chromosomes.Micelacking
boththeH2AXandp53proteins arehighly pronetoboth
hematopoieticandsolidtumors.Evenmicelackingone copyoftheH2AX
geneinthecontextofp53deficiency show significantly increased rates
of lymphomas. Theseresponsesillustratehowhighlycomplexthe DNArepair
processis, and how defectsin any of
itsindividualcomponents-manystillunidentified-openthe
doortotheappearanceofcancer.Provocatively,the human H2AX gene maps
toa chromosomal region (llq23)
thatfrequentlyundergoeslossofheterozygosityandlor
deletion,creatingthepossibilitythatthisgeneand
encodedhistonearefrequentlyshedby humancellsen route to malignancy.
Figure12.41y-H2AX and double-strandDNA breaks The creationof dsDNA
breaksbyvarious mechanismsresultsinthe phosphorylationof
H2AXhistone (avariant formof histone H2A),yieldingy-H2AX. Li keH2A,
H2AX participatesinthe form