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