Karran P Nature Review

8/2/2019 Karran P Nature Review

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Th thipurins azathiprin, 6-thiguanin (6-TG)and 6-mrcaptpurin (6-MP) ar ffctiv anti-inflammatr, anticancr and immunsupprssivdrugs. Th hav bn availabl in clinical practicfr vr half a cntur, with 6-MP and azathiprinrciving US Fd and Drug Administratin (FDA)apprval in 1953 and 1968, rspctivl1. Th arnafr thipurin mdicatin has significantl divrsifidsinc thir intrductin and bth th n- and th ff-labl applicatins fr inflammatr and autimmundisass ar xtnsiv.

Th Intrnatinal Agnc fr Rsarch n Cancr(IARC) has, hwvr, dtrmind that thr ar suf-ficint grunds t classif azathiprin as a humancarcingn2, and it is nw wll rcgnizd that pr-lngd tratmnt with thipurins is assciatd withan incrasd risk f varius malignancis. In supprt

f this cnclusin, IARC cits th incrasd inci-dnc f nn-Hdgkin lmphma, skin squamus cllcarcinma (SCC), hpatbiliar carcinma and ms-nchmal tumurs in rnal transplant patints. ThIARC rprt als nts that th sam malignancis armr frqunt albit t a lssr xtnt in patintstratd with azathiprin fr, inter alia, rhumatidarthritis, sstmic lupus and inflammatr bwl dis-as (s sctin n chrnic inflammatr disrdrs).Much f th incrasd cancr in transplant patints canb attributd t ncgnic viruss, prbabl as a cn-squnc f immunsupprssin, and is nt dirctlrlatd t th mchanism f actin f azathiprin.

Th imprtant cntributin f immunsupprssin isfurthr highlightd b th parallls btwn th rangf cancrs in transplant rcipints and amng HIV-infctd individuals3,4. Kapsi sarcma, nn-Hdgkinlmphma, livr cancr and crvical cancr ar fr-qunt in bth grups. Amng transplant patints, thpst-transplant lmphprlifrativ disrdrs, whichinclud nn-Hdgkin lmphma, ar als likl t bf viral rigin. Ths malignancis, which ar scndnl t skin SCC in frqunc, ar prdminantlarl-nst and ccur mstl within th first araftr transplant. A pssibl viral atilg fr SCC intransplant patints has nt bn xcludd, althughvidnc fr a causativ assciatin with knwnncgnic human papillmavirus tps has prvdlusiv5. It rmains pssibl thrfr, that sm f thcancr assciatd with azathiprin tratmnt rflcts

th prprtis f 6-TG incrpratd int DNA. DNA6-TG can b cnsidrd th ultimat activ mtablitf all th thipurins. 6-TG accumulatin in DNAis an invitabl cnsqunc f thipurin tratmntand might b cntral t thir thraputic ffcts. Wdscrib sm f th chmical prprtis f DNA6-TG and xplr, in brad trms, hw ths mightacclrat carcingnsis. As clinical xampls, wwill cnsidr mldsplastic sndrm-assciatdacut mlid lukamia (AML) and skin cancrin rgan-transplant rcipints. W als addrss thptntial cnsquncs f lng-trm thipurintratmnt fr chrnic inf lammatr cnditins.

Cancer Research UK London

Research Institute, Clare Hall

Laboratories, South Mimms,

Hertfordshire, EN6 3LD, UK.

Correspondace to P. K.

e-mail:

[email protected]

doi:10.1038/nrc2292

Thiopurines in current medicalpractice: molecular mechanisms andcontributions to therapy-related cancerPeter Karran and Natalie Attard

Abstract | Thiopurines have diverse clinical applications and their long-term use as anti-

rejection drugs in transplant patients has been associated with a significantly increased

risk of various types of cancer. Although they are slowly being replaced by a newgeneration of non-thiopurine immunosuppressants, it is anticipated that their use in the

management of inflammatory and autoimmune diseases will continue to increase.

Therapy-related cancer will remain a potential consequence of prolonged treatment for

these generally non-life-threatening conditions. Understanding how thiopurines

contribute to the development of cancer will facilitate clinical decisions about the

potential risks to patients of long-term treatment for chronic inflammatory disorders.

R E V I E W S

24 | jANUARy 2008 | VoLUMe 8 www.tu.om/iws/
mailto:[email protected]:[email protected]


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Therapeutic indications for thiopurine treatment

Th principal thraputic ffcts f thipurins canb dividd int tw brad clinical catgris: antican-cr and immunsupprssiv. Thir pharmaclgicalmd f actin will b dscribd in dtail in th nxtsctin.

Anticancer. Initiall dvlpd as an antagnist f purinutilizatin, 6-MP was shwn t inhibit tumur grwth inrdnts6. Th pinring us f 6-MP as a tratmnt fracut lmphblastic lukamia7, th cmmnst child-hd cancr, cntributd t a dramatic imprvmnt insurvival frm this malignanc, which prviusl had adismal prgnsis.

Immunosuppression. Shrtl aftr its dvlpmnt, 6-MPwas shwn t hav immunsupprssiv prprtis. on f

th utstanding succsss f th thipurins, principallazathiprin, sinc thir intrductin in th 1960s is thircntributin t incrasd graft survival fllwing rgantransplant. In cmbinatin with crticstrids, thdminatd this fild fr narl tw dcads. Azathiprinis incrasingl bing rplacd in this cntxt b th nwrgnratin f immunsupprssants such as mcphn-lat mftil, tacrlimus and rapamcin (s FIG. 1a) andth xtnt t which azathiprin has bn phasd ut asan immunsupprssant in rnal transplant patints vrth past dcad is shwn in FIG. 1b. In particular, b 2003mcphnlat mftil a prdrug f mcphnlicacid, which is an inhibitr f insin mnphsphat

dhdrgnas (s th fllwing sctin) was prscribdfr apprximatl 80% f rnal transplant patints in thUnitd Stats at th tim f discharg frm hspital. Thwisdm f th wrldwid rplacmnt f azathiprinb mcphnlat mftil is brught int qustin b arcnt stud in which rnal transplant patints rciv-ing th nwr micrmulsin ciclsprin prparatin incmbinatin with ithr azathiprin r mcphnlatmftil achivd cmparabl lng-trm utcms in trmsf graft survival8. In additin, th avrag cst pr patintis 15-fld highr fr mcphnlat mftil than fr aza-thiprin, raising qustins abut th cst-ffctivnss fpatint car9. In viw f thir rlativl rcnt intrductinint rutin clinical practic, whthr ths nvl immu-nsupprssants cntribut t pst-transplant malignancrmains an pn qustin10.

Th immunsupprssiv prprtis f thipurins

ar xplitd in th managmnt f a numbr f chrnicinflammatr and autimmun disass, includingrhumatid arthritis, sstmic lupus rthmatsus,pmphigus vulgaris, drmatmsitis, multipl scl-rsis and masthnia gravis. In this catgr, prbablth mst lng-standing xprinc has bn gaind ininflammatr bwl disas (IBD) fr which thipurinshav bn usd sinc th 1960s11.

Thipurin us rmains widsprad and, in ffct,dctrs in mst hspital-basd mdical spcialitis willlk aftr patints wh ar taking r hav takn thipu-rin drugs at sm pint. Thir acut lif-thratningsid ffcts ar wll knwn12. It is quall imprtant

At a glance

Thethiopurinesazathioprine,6-mercaptopurineand6-thioguanine(6-TG)havebeenavailabletomedical

practitionersforoverhalfacentury.Theyareusedasanticancerandimmunosuppressiveagents.Theintroductionof

azathioprineasanimmunosuppressantrevolutionizedsolid-organtransplantationfromunrelateddonorsand

resultedinmuchimprovedgraftsurvival.Thethiopurinesarerecognizedtreatmentoptionsforanincreasingnumber

ofchronicinflammatoryandautoimmunedisorders,includingarthritisandcolitis.

Largelyonthebasisofepidemiologicaldataofcancerintransplantpatients,theInternationalAgencyforResearchon

Cancerclassifiesazathioprineasahumancarcinogen.Muchofthisincreasedcancercanbeattributedtotheeffectsofimmunosuppressionandtheinvolvementofoncogenicviruses.Insomecases,however,demonstrationofaviral

aetiologyhasprovedelusive.Thisisparticularlytrueofskincancer,whichisthemajortreatment-relatedcancer

amongtransplantpatients.

Thiopurinesareprodrugsandoneoutcomeoftheircomplexmetabolismistheincorporationof6-TGintoDNAduring

replication.6-TGischemicallymorereactivethancanonicalDNAbasesandundergoesmethylationin situ inDNA.

MethylatedDNA6-TGisultimatelycytotoxicbyamechanismthatdependsonthecellsDNAmismatchrepairsystem.

Onerouteofescapefromthecytotoxicityofthiopurinesisbyinactivationofmismatchrepair.Mismatchrepair

defectsareassociatedwithhighratesofspontaneousmutationandarecommonincertaintypesofcancer.Acute

myeloidleukaemiaoccursmorefrequentlythanexpectedintransplantpatients.Theseazathioprine-relatedcancers

areoftendefectiveinmismatchrepair.

DNA6-TGisalsophotochemicallyreactiveandhasamaximumabsorbanceat340nmintheUVAregionofthe

ultravioletspectrum.UVAcomprisesmorethan90%ofsolarradiationthatreachestheearthand,onexposureto

UVA,the6-TGDNAchromophoregeneratesreactiveoxygenspecies(ROS),whichcandamageDNA,proteinsand

othercellularmacromolecules.DNA6-TGitselfisparticularlysusceptibletooxidationbyROStoformguanine-6-sulphonate.Thisphotoproductisa

powerfulblocktoreplicationbutcanbebypassedbyY-familypolymeraseswhichhavearelativelyrelaxedstringency.

ThephotochemicalreactionsofDNA6-TGaremutagenicandthismightcontributetoanincreasedriskoftransplant-

relatedsquamouscellcarcinomaoftheskin.

Theassociationofazathioprinewiththerapy-relatedcancersanditsincreasinguseintreatmentofchronic

inflammatoryandautoimmunedisorderssuggeststhatcarefulmonitoringofthesepatientsforsignsofpossible

therapy-relatedcancerisadvisable.

R E V I E W S

NATURe ReVIeWS |cancer VoLUMe 8 | jANUARy 2008 |25


3/13

19940

20

40

60

80

100

0

20

40

60

80

100

%p

atients

Year1995 1996 1997 1998 1999 2000 2001 2002 2003 1994

Year1995 1996 1997 1998 1999 2000 2001 2002 2003

b

a

Aza MMF Rapamycin Ciclosporin Tacrolimus

Calcineurin inhibitors:

Ciclosporin

Tacrolimus

Disrupt cytokine gene expression

by NFAT inhibition

mTOR inhibitors:

Rapamycin (sirolimus)

Everolimus

Inhibit serine/threonine protein

kinase activity of mTOR

Antimetabolite:

Aza

1960s 1980s 1990s

Glucocorticoids:

Prednisolone

Methylprednisolone

Antimetabolite:

MMF

Inhibits de novo purine synthesis

by IMPDH inhibition

Purine salvage pathway

Clls can obtain th purinbass thy nd to form th

prcursors of DNA and RNA

ithr by synthsizing thm

de novo or by rcycling from

dgradd nuclic acids

through this pathway.

Myelosuppression

A condition in which th

production of blood clls by

th bon marrow is

significantly rducd. This can

rsult in anamia, lif-

thratning infction and

spontanous blding.

that th ptntial carcingnic cnsquncs f thirprlngd us ar als apprciatd. Undrstandinghw ths drugs ar carcingnic culd rsult in thsdistrssing sid ffcts bing minimizd r liminatd.

Metabolism, immunosuppression and cytotoxicity

Thipurins ar prdrugs and nd mtablic cnvr-sin int activ cmpunds13, althugh th cntributinf varius activ mtablits t thir pharmaclgicalffcts has nt bn unquivcall dfind. Th firststp f azathiprin activatin is th rmval f thsubstitutd imidazl ring in a nn-nzmatic rac-tin invlving glutathin. This ccurs in rthrctswhich thn rlas 6-MP, th activ mtablit. As with6-TG, 6-MP is radil transprtd int clls14, whr

it ntrs th purin salvag pathway. Bth thipurinsar gd substrats fr hpxanthinguanin phs-phribsltransfras 1 (HPRT1), th first nzmaticstp f salvag. HPRT catalss th additin f ribs5-phsphat t gnrat thiinsin mnphsphat(TIMP) and thiguansin mnphsphat (TGMP)frm 6-MP and 6-TG, rspctivl(FIG. 2). This is thfirst stp in thir cnvrsin t thiguanin nucltids(TGNs), which ar prcursrs fr th incrpratin f6-TG int RNA and DNA. In cmpting ractins, thdgradatin f intracllular 6-MP is initiatd b xan-thin xidas and bth 6-MP and 6-TG ar inactivatdb thipurin S-mthltransfras (TPMT)15.

Th nrmal functin f human TPMT, which iscnsrvd amng spcis frm bactria t mammals16,is unknwn. Th bactrial hmlgus cnvrt sl-nium- and tllurium-cntaining cmpunds t lss txicmthlatd drivativs using S-adnslmthinin(SAM) th surc f mthl grups fr numrusnzm ractins as a mthl grup dnr17. Thr isspculatin that th human nzm might als dtxifths mtablicall imprtant, but ptntiall pisn-us, mtals16. Th bst charactrizd activit f humanTPMT is, hwvr, th dtxificatin f 6-MP and 6-TGwhich, tgthr with TIMP and TGMP18, ar cnvrtdt inactiv mthlatd prducts. Mthlatin f all thsintrmdiats impairs th snthsis f TGNs and thrbrducs th ffctiv thipurin ds. Th significanc

f this catablism is mphasizd b th bsrvatin thatth


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Me6-TG

HN

N NH

N

S

6-MP

O

OHOH

HN

N N

N

S

P

HO

TXMP

O

OHOH

HN

N N

N

S

P

H2N

TGMP

O

OH

HN

N N

N

S

P

H2N

Thio-dGTP

PP

O

OHOH

HN

N N

N

S

P

H2N

MeTGMP

CH3

O

OHOH

HN

N N

N

S

P

MeTIMP

CH3

O

OHOH

HN

N N

N

S

P

TIMP

Me6-MP 6-Thiouric acid

TPMT XO

HPRT

HPRT IMPDH GMPS

TPMT

TPMTTPMT

Kinases

Reductase

HN

N NH

N

S

Azathioprine

N

NO2N

CH3

HN

N NH

N

H2N

S

6-TG

Non-enzymatic removalof nitroimidazole group

Inhibitor ofde novopurine biosynthesis

Replicative DNApolymerase substrate

Km

Drivd from nzym kintics,

Km

is th substrat

concntration at which an

nzymatic raction procds

at half-maximal vlocity. It is

ffctivly a masur of th

affinity of an nzym for a

particular substrat.

Km

similar t that f unmdifid dGTP. 6-TG is ntknwn t b subct t prrplicativ xcisin rpairand can accumulat in DNA. Rplicatin f a 6-TGin th tmplat DNA strand is als rlativl as andth thipurin is nt a mar bstacl t lngatin bDNA plmrass in simpl primr xtnsin assas21,22.Cding b 6-TG is ambiguus, hwvr, and C and Tar incrpratd with apprximatl qual facilit23. Inintact clls, a lw lvl f DNA substitutin b 6-TG isnithr txic nr particularl mutagnic. Highr lvls

induc significant txicit that is abslutl dpndntn activ purin salvag. As clls hav nl a singl activcp f th X-linkd HPRT1 gn that ncds th knzm in th salvag pathwa, th acquir rsistanc thigh cncntratins f 6-TG b a singl mutatinal vntthat inactivats HPRT. This slctin fr 6-TG rsistancis th basis f th widl usd HPRT mutatin assa inculturd human clls.

Thipurin txicit is a dlad ffct. This partlrflcts th rquirmnt fr passag thrugh nS phas f th cll ccl t allw incrpratin f 6-TGint DNA. In fact, cttxicit is dlad vn lngrthan this, suggsting a rquirmnt fr passag thrugh

at last n mr S phas t allw rplicatin f th6-TG substitutd DNA24,25. This xtnsiv dla, andth invlvmnt f rplicatin f th mdifid DNA,is an unusual prprt shard with mthlating agntsthat prduc DNA O6-mthlguanin (O6-mG)2627 a DNA lsin that is structurall similar t 6-TG.Th bsrvatin that clls culd dvlp simultan-us rsistanc, knwn as mthlatin tlranc, tmthlating agnts and t thipurins suggstdthat th cttxic ffcts f ths tw vr diffr-

nt classs f drug invlv a cmmn mchanism28rlatd t th similarit btwn O6-mG and 6-TG.Mthlatin tlranc was shwn t b gnticallrcssiv29,30, cnsistnt with an scap frm clldath b lss r inactivatin f a functin rquird tprcss bth DNA 6-TG and DNA O6-mG. earlirxprimnts in bactria31 indicatd that th DNAmismatch rpair sstm (MMR) was a likl suspctfr prcssing O6-mG-cntaining, and b xtnsin,6-TG-cntaining, bas pairs that shar sm f th g-mtrical prprtis fbona fide mismatchs32(FIG. 3).Prcssing causd cll dath, and txicit was allviatdb mutatins that disabld MMR.

Figure 2 | Tiouis d ti mtboism. Azathioprine is converted to 6-mercaptopurine (6-MP) by non-enzymatic

activation in red blood cells. Both 6-MP and 6-thioguanine (6-TG) are salvaged by the hypoxanthineguanine phosphori-

bosyltransferase (HPRT) of immune effector cells and converted into their respective nucleoside monophosphates (TIMP

and TGMP). In competing catabolic reactions, thiopurine S-methyltransferase (TPMT) inactivates 6-MP and 6-TG by

S-methylation and xanthine oxidase (XO) converts 6-MP to 6-thiouric acid. TIMP and TGMP are also TPMT substrates.

Methylated TIMP (meTIMP), but not meTGMP, is an effective inhibitor of de novo purine biosynthesis. TIMP that escapes

catabolism is further metabolizedbyinosine monophosphate dehydrogenase (IMPDH) and guanine monophosphate

synthetase (GMPS) to TGMP. Sequential action of deoxynucleoside kinases and reductase generates the thioGTP andthio-dGTP that are the substrates for incorporation of 6-TG into RNA and DNA.

R E V I E W S

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=3251http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=3251


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T link activ MMR t cll dath in human clls, amdl was prpsd28 in which pst-rplicativ prcss-ing b MMR f abrrant bas pairs cntaining 6-TG rO6-mG gnratd ptntiall lthal DNA lsins bcausth abrrant bas was in th tmplat (FIG. 3b), rathr thanin th daughtr DNA strand, which MMR nrmall dits(FIG. 3a). Prcssing f ths particular bas pairs wasinvitabl futil bcaus DNA plmrass culd nvrfind a sufficintl prcis daughtr-strand partnr frth rgu bas that wuld allw it t scap th attn-tin f MMR. Th rplicatin frk is, manwhil, frt advanc laving ths unrslvd prcssing attmptsin its wak.

Sm f th mlcular vnts undrling th dladcll-ccl ffcts that ar assciatd with MMR-dpndntDNA damag intractins hav bn dfind andths prvid supprt fr th futil prcssing mdl.Mthlating agnts r 6-TG prvk a dlad MMR-dpndnt G2 cll-ccl chckpint25,33,34. Carful analsisrvals that MMR-dpndnt G2 chckpint activatinis mdiatd bATR (ataxia tlangictasia and Rad3-

rlatd)ATRIP (ATR intracting prtin) and CHeK1,which ar assciatd with abrrant vnts at rplicatinfrks34,35. Th ATM DNA damag snsr is nt rquirdfr this DNA damag rspns but is activatd in a MMR-indpndnt mannr b DNA mthlatin damag thrthan O6-mG36,37. ATR and CHeK1 activatin dpndsn MutS (an MSH2 and MSH6 dimr) and MutL (adimr fMLH1 and PMS2) and it sms likl that thlat triggring f th chckpint is causd b anmalusDNA structurs38 gnratd during rplicatin thrughprsistnt unrslvd MMR intrmdiats frm thprvius cll ccl. on f th mst rcnt bichmi-cal studis prvidd dirct vidnc fr th rptitiv

prcssing fO6-mG:T bas pairs b MMR in cllxtracts39. Dirct assciatin f ATRATRIP with MutSand MutL35, r th rcruitmnt f appttic signallingfactrs at MutS bund t th abrrant bas pairs40, havals bn prpsd as mchanisms b which cllular sig-nalling r apptsis might b initiatd withut th ndfr mismatch crrctin attmpts. ovrall, th timingf th DNA-damag-chckpint rspns supprts thfutil prcssing cncpt and mst f th vidnc sug-gsts that bas pairs cntaining O6-mG r 6-TG d ntdirctl prvk G2 arrst r cll dath. Th ptntialllthal DNA damag that ffcts chckpint activatinis causd b rplicatin thrugh rgins f unrslvdMMR attmpts gnratd during th prvius S phas.

Th clls that undrg ATRCHeK1-rlatd G2arrst aftr th scnd S phas fllwing damag almstcrtainl cntain abrrant DNA structurs that hav ahigh prbabilit f bing lthal and can b rpaird brcmbinatin. Mthlating agnts and 6-TG ar assci-atd with charactristic MMR-dpndnt rcmbinatinvnts41,42. It fllws frm this that inactivatin f MMR,

b prvnting th frmatin f ptntiall lthal dubl-strand braks, incrass cll survival. This tlrancf mthlatin r 6-TG-rlatd DNA damag cmsat th cst f a significantl incrasd rat f spnta-nus mutatin du t uncrrctd rplicatin rrrs.Th liklihd f a cll acquiring mutatins that allwit t scap frm th nrmal cnstraints n grwth isthrb incrasd and th tim rquird fr malignanct dvlp is rducd. B wa f illustratin, inhritdmutatins in gns ncding cmpnnts f MutS rMutL caus arl-nst cancr in ppl with hrditarnnplpsis clrctal cancr sndrm43.

Althugh th riginal futil prcssing mdl pr-vidd a satisfing qualitativ xplanatin fr th invlv-mnt f MMR in th txicit f mthlating agnts andthipurins, thr wr sm quantitativ incnsistncis.equitxic tratmnts wr assciatd with apprximatl1,000 DNA O6-mG lsins but as man as 107 DNA 6-TGs. This puzzling discrpanc was rslvd b Swannand his cllagus44, wh dmnstratd that th baspairs that prvk MMR invlv a mthlatd frm fDNA 6-TG (m6-TG) that is gnratd b an in situ, nn-nzmatic ractin f DNA 6-TG with SAM, which isa wak mthlating agnt. onl ~1 in 104 DNA 6-TGsundrgs mthlatin. Thus, whn clls ar tratd withthipurins r mthlating drugs, similar numbrs fDNA m6-TG (thrugh SAM mthlatin) and O6-mG

(frm dirct mthlatin) lsins caus similar xtnts fcll killing. SAM-mdiatd chmical mthlatin f DNA6-TG prvids a striking xampl f th incrasd ractiv-it that accmpanis rplacmnt f th guanin xgnatm b sulphur45. Mthlatin f th o6 atm f guaninb SAM is unfavurabl and wuld nl gnrat apprxi-matl 10 DNA O6-mGs during a tpical S phas46. DNA6-TG is much mr ractiv and th rat f S-mthlatinis narl tw rdrs f magnitud highr than that f gua-nin whn clls ar tratd with wak mthlating drugs47.This nhancd ractivit mans that DNA 6-TG bcmsmthlatd t a significant xtnt, vn at rlativl lwintracllular cncntratins f SAM.

Box 1 | Thiopurine S-methyltransferase testing in the clinic

ThevalueofthiopurineS-methyltransferase(TPMT)testingbeforeinitiationof

azathioprineor6-MPtherapyiscontentious.Allelesforlow-andhigh-levelTPMT

activityareinheritedinanautosomalco-dominantmanner98resultinginthreedistinct

phenotypes:normal/high,intermediateanddeficientS-methylators,comprising

approximately89%,11%and0.3%ofthepopulation,respectively.Themostcommon

variantallelesencodinglowTPMTaredesignatedTPMT*3AandTPMT*3C.The1in300

peoplewhoinheritacombinationoftwolow-activityTPMTallelesaredeficientinthiopurinemethylationandsuffersevereandearlymyelotoxicityaftertaking

azathioprineor6-MP.Theclinicalrationaleforpriortestingistoidentifytheserare

instancesofTPMTenzymedeficiencyandconsequentlyeitheravoidazathioprineand

6-MPentirelyorprescribeatamuchlowerdosewithintensifiedmonitoringforpossible

toxicsideeffects.Additionalaimsaredosemodificationtoreducetoxicityinpatients

withanintermediatephenotypeandtoavoidsub-optimaltreatmentofpatientswith

normalorhighenzymeactivity.TheadditionalclinicalbenefitthatidentifyingTPMT

statushasovertraditionalmonitoringofclinicalandserologicalparametersisnotclear,

anditisuncertainwhetherthisknowledgecanbeusedtoindividualizeadosing

regimen.Mercaptopurinemetabolismiscomplexandmaybeinfluencedbyfactors

suchasunderlyingdisease,co-medicationandrace99.Furthermore,TPMTstatusisnot

theonlydeterminantofmyelosuppression,whichremainsapotentialconsequence

eveninpatientswithnormalTPMTactivity.Theneedforclosemonitoringofblood

countsisnotnegatedinthesepatientsandpublishedreportsofcost-effectivenessof

routineTPMTtestingareconflicting100,101

.Despitethis,priorTPMTtestingisrecommendedbytheFoodandDrugsAdministrationintheUSAandisrecognizedas

bestpracticeinauthoritativeguidelinesindermatology102.Itsusefulnessremains

unsupportedbyfirmevidence.

R E V I E W S

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6/13

G

a

b

Misincorporation

Misincorporation

Polymerase-associatedPCNA sliding clamp

Daughter strandof DNA

Excision and resynthesis

Reiterativeprocessing

Recognitionand processing

G

3

5T

X

3

5T

DNA polymerase

MutS

Recognitionand processing

3

5

EXO1

PolPCNA

Aberrant recombinogenicDNA structure

Excision andresynthesis

EXO1PolPCNA

C

5

5

X5

5

G

3

5T

MutL

X

3

5T

G2 arrest

ATRATRIP

CHEK1

X = me6-TG orO6-meG

Similarl t 6-TG, DNA m6-TG cds ambigu-usl during rplicatin and dircts C and T insrtinapprximatl quall44. DNA m6-TG:T bas pairsar rcgnizd particularl wll, and significantl bt-tr than 6-TG-cntaining pairs, b MutS. This is fullcnsistnt with thir prcssing b MMR int ptnt,ptntiall lthal DNA lsins.

on imprtant implicatin f ths findings is thatth rsistanc f MMR-dficint clls prvids thmwith a significant prlifrativ advantag vr thr-wis idntical MMR prficint clls whn challngd ba thipurin r mthlating agnt. In labratr studis,

chrnic xpsur t scalating cncntratins f ithrtp f drug prmits th slctin f rar MMR-dfctiv

variants frm a ppulatin f MMR-prficint clls29,48,49.This prprt ma b imprtant in th dvlpmnt fcancr in patints rciving thipurins (s blw).

Th nhancd rsistanc t hamatlgical txicitand dath in MMR-dficintMsh2/ knckut mictratd with 6-MP is lqunt tstimn t th dirctinvlvmnt f MMR in th bilgical ffcts f thi-purins. Althugh inactivatin f MMR is undubtdlamng th mst imprtant mchanisms b which cllsacquir thipurin rsistanc, it is b n mans th nl

Figure 3 | Dna mismt i d its otibutio to ottiy t Dna dmg. | Correction of

replication errors. Erroneous incorporation of a non-complementary base by the replication apparatus (DNA

polymerase plus the polymerase-associated proliferating cell nuclear antigen (PCNA) sliding clamp triggersrecruitment of the mismatch recognition factor MutS (an MSH2 and MSH6 dimer). A second dedicated MMR factorMutL (a dimer of MLH1 and PMS2) is then recruited. The endonuclease activity of MutL introduces nicks that

promote the loading of EXO1 on the 5 side of the misincorporated nucleotide. The 5-to-3 exonucleolytic activity ofEXO1 excises a stretch of daughter strand of DNA including the mismatch. The extensive gap left by EXO1 is filled by

DNA polymerase PCNA, which now incorporates the correct complementary nucleotide, and mismatch repair iscompleted by DNA ligase sealing the remaining nick. The key to successful correction is that the incorrect nucleotide

resides, by definition, in the newly synthesized daughter DNA strand. b | The presence of a miscoding base analogue

(X, for example, methyl 6-thioguanine (me6-TG) or O6-meG) in the template strand can confuse the MMR system. Here

me6-TG derived by chemical S-methylation of 6-TG incorporated in an earlier round of replication, or O6-meG

produced by a methylating agent, directs the incorporation of T. Me6-TG:T or O6-meG:T base pairs do not escape

surveillance by MutSMutL, which triggers mismatch correction attempts that remain incomplete because of theimpossibility of incorporating a perfectly paired base opposite the lesion28, 31. The anomalous DNA structures generated

by the incomplete repair attempts can be observed as DNA strand interruptions34. During the S phase of the next cell

cycle, these are converted into potentially lethal aberrant DNA structures that are substrates for recombination. These

trigger activation of ATR (ataxia telangiectasia and Rad3-related)ATRIP (ATR-interacting protein) DNA-damage

signalling, phosphorylation of CHEK1 and G2 cell-cycle arrest.

R E V I E W S



7/13

n. Altratins in TPMT r HPRT activit thipurintransprt int clls50 ar als ptntial cntributrs.Whthr th rcntl dscribd intractin btwnDNA cntaining 6-TG:T (ReF. 51) r 6-TG:C (ReF. 52)and an apparnt multiprtin cmplx cntainingglcraldhd-3-phsphat dhdrgnas has an sig-nificant rl in dtrmining th txicit f thipurins iscurrntl uncrtain.

Purine biosynthesis. Thr is sm vidnc t suggstthat th abilit f 6-MP mtablits t inhibit de novopurin bisnthsis ma cntribut t thir cttxicffcts. In additin t th fr thipurin bass, TPMTals mthlats bth TIMP and TGMP18(FIG. 2). Thprduct f th frmr ractin, mthlthiinsin mn-phsphat (mTIMP) is an fficint nn-cmptitivinhibitr f phsphribslprphsphat amidtrans-fras (PPAT)53, which catalss an arl stp in th path-wa f purin bisnthsis. Inhibitin rducs supplis fpurin triphsphats fr DNA and RNA snthsis, cllularmtablism and signalling, and is thught t cmprmis

th clnal xpansin f T clls. Dspit arl suggstinst th cntrar24, th cttxic prprtis f thipurinsar still ftn thught t b a cnsqunc f th dpltinf purin nucltids and thir immunsupprssiv ffctsar thught t rflct a particular rlianc f T clls n thde novo purin snthsis rathr than salvag. It shuld bntd, hwvr, that ffctiv salvag is an ssntial firststp in thipurin mtablism that prcds th frma-tin f mTIMP. It is far frm clar that PPAT inhibitinis a mar cntributr t thipurin-inducd cttxicitand immunsupprssin.

Th xtrm mltxicit f thipurins in patintsprvids th mst dirct vidnc that mTIMP frma-tin ds nt accunt fr thir cttxic prprtis.Mltxicit is assciatd with lw, rathr than high,TPMT and mTIMP lvls54,55. Incrasd accumulatinf 6-TG in DNA and RNA as a rsult f high TGN lvlsdu t inadquat thipurin catablism prvids a mrlikl xplanatin fr txicit. Indd, th rlatinshipbtwn TGN lvls and mlsupprssin has bnstrssd man tims56,57.

If th immunsupprssiv ffcts f thipurinsrall ar a cnsqunc f rducd de novo purinsnthsis, it fllws that thipurins must xrt thircttxic and immunsupprssiv ffcts thrugh dif-frnt mchanisms. Is it in fact ncssar t pstulattw mchanisms r might immunsupprssin als b

du t accumulatin f TGNs and, ultimatl, accumu-latin f 6-TG in DNA? Pwrful inhibitin fde novopurin snthsis is crtainl immunsupprssiv. Thisis xmplifid b mcphnlat mftil, a mrrcntl dvlpd immunsupprssant that is incras-ingl rplacing azathiprin (FIG. 1). Mcphnlatmftil is mtablizd t mcphnlic acid (MPA)b plasma strass. MPA is a pwrful and slctivinhibitr f insin mnphsphat dhdrgnas(IMPDH), which catalss th rat-limiting stp fGTP snthsis, and dplts th lvls f GTP anddGTP58. It ds nt fllw frm this, hwvr, thatmTIMP inhibitin f th nn-rat-limiting PPAT in

th sam pathwa undrlis immunsupprssin b6-MP and azathiprin.

As thipurins hav bn xtrml succssful in thtratmnt f childhd lukamia, man f th in vitrostudis which implicatd IMPDH inhibitin b 6-MPmtablits wr carrid ut with stablishd tumurcll lins f T-cll rigin. Unfrtunatl, man f th clllins usd in ths studis hav sinc bn shwn t bdfctiv in DNA mismatch rpair. Ths includ thjurkat (MutS hmlgu 2 (MSH2)-dficint)59, CCF-CeM (MutL hmlgu 1 (MLH1)-dficint)60, 293T(MLH1-dficint)61 and MoLT4 (PMS2-dficint)62cll lins, all f which ar atpicall thipurin-rsistantbcaus th lack th mst significant xcutinr anactiv MMR sstm63. Thus, althugh vrxprssin fa transfctd TPMT in CCF-CeM clls inducd >99%inhibitin fde novo purin snthsis, th changs in6-TG and 6-MP snsitivit wr small64. This suggststhat th mst significant mchanism f cttxicit iscmmn t bth thipurins, invlvs TGN frmatinand 6-TG incrpratin int DNA, and rquirs activ

mismatch rpair.Clinical findings d nt supprt a prdminant rl

fr de novo purin snthsis inhibitin in cttxicit.Drviux et al.65 shwd that th rat fde novo purinsnthsis in th T lukamic clls f patints wasunchangd b succssful 6-MP tratmnt. B cntrast,mthtrxat, an acknwldgd and ptnt inhibitrf nucltid bisnthsis, was an xtrml ffctivinhibitr and significantl nhancd th thraputicffct f 6-MP. This suggsts that 6-MP cttxic-it twards lukamic T clls is largl indpndntf an ffct n de novo purin snthsis. Findingsfrm mus mdls supprt this cnclusin. 6-MPtratmnt causs significantl lss mltxicit inMMR-dfctivMsh2/ knckut mic than in thirrpair prficintMsh2+/+ rMsh2+/ cuntrparts. Thphntp f th TPMT knckut mus66 prvidsmr vidnc that 6-MP and 6-TG shar a cmmnmchanism f txicit. Dspit prducing n masur-abl mTIMP, Tpmt/ animals ar much mr snsitivt 6-MP than hmzgus wild-tp r htrzgusmic, in which mTIMP lvls ar mr than 1,000-fld highr. Thr sms littl dubt that th txicand mlsupprssiv prprtis f 6-MP and 6-TGrflct th frmatin f TGNs and 6-TG incrpratinint nuclic acids. Althugh th immun status f thanimals was nt addrssd dirctl, thr sms littl

rasn t invk an altrnativ t TGNs t xplain thimmunsupprssiv prprtis f th thipurins.

DNA 6-TG and UVA radiation

Th mthlatin in situ f DNA 6-TG dscribd abvis n xampl f its nhancd chmical ractivit. Itsintractin with ultravilt (UV) light prvids a scndxampl. Th cannical DNA and RNA bass all absrbmaximall in th UVC spctral rgin (~260 nm) andtrrstrial lif basd n th cnvntinal nuclic acids ispssibl nl bcaus rmval f UVC frm incidnt sun-light cnsidrabl rducs th thrat f dstructiv ph-tchmical damag t DNA. Thipurins, b cntrast,

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8/13

0.00

0.00

250 300 350 400

0.02

0.04

0.06

0.08

0.10

0.12

0.14

UVC (100280) UVB UVA (320400)

Wavelength (nm)

Absorban

ce

G

6-TG

HN

N NH

N

H2N

S

6-TG

Chromophore

That part of a substanc that

absorbs visibl light or, by

xtnsion, ultraviolt radiation.

Reactive oxygen species

(ROS). Highly unstabl oxygn-

containing chmical ntitis.

ROS includ oxygn fr

radicals such as th hydroxyl,

proxyl and suproxid anion

radicals and non-fr radical

forms such as singlt oxygn.

Low-lvl ROS production is an

ssntial componnt of

intracllular signalling.

Fenton-like reactions

Many of th most harmful

changs in DNA ar causd by

hydroxyl radicals. In th

Fnton raction, th rlativly

innocuous hydrogn proxid

is convrtd into hydroxyl

radicals in a raction involving

iron associatd with DNA.

Tm

Th tmpratur at which a

DNA doubl hlix dissociats

into singl strands.

ar significant UVA chromophorsand bth 6-TG and6-MP hav an absrbanc maximum at apprximatl340 nm (FIG. 4). As UVA cmpriss vr 90% f slarultravilt radiatin that rachs earth, 6-TG in DNAis a ptntial hazard. Bcaus f its lngr wavlngths,UVA radiatin is nt ffctivl liminatd b glass andit als pntrats significantl dpr int skin. As muchas 2030% f UVA incidnt at th skin surfac pntratst th lwr lars that cntain th stm clls rspnsi-bl fr skin rnwal67. Fr a gnral rviw f th halthaspcts f ultravilt radiatin, s ReF. 68.

In aquus slutin, bth 6-TG69 and 6-MP70 arTp I and Tp II UVA phtsnsitizrs that gnrathighl damaging ractiv oxygn spcis(RoS) whn irra-diatd in th prsnc f mlcular xgn. In a Tp Iractin, th thipurin absrbs UVA nrg and is cn-

vrtd t an unstabl xcitd triplt stat that can intractwith xgn t frm a thipurin radical and suprxid(o

2). Fnton-lik ractions cnvrt o

2int th hdrxl

radicals (oH) that ar a surc f damag t cllular

macrmlculs. Tp II ractins invlv transfr fth absrbd radiatin nrg dirctl t grund-stattriplt mlcular xgn (3o

2) t gnrat singlt xgn

(1o2) a nn-radical with a lngr half-lif than mst

RoS. Bth oH and 1o2caus ptntiall mutagnic

damag t DNA. This includs brakag f th DNAstrands as wll as xidatin-inducd changs t cnstitu-nt bass and dxribs. 1o

2is gnrall cnsidrd t

b th prdminant damaging spcis prducd in cllsxpsd t rlativl high fluncs f UVA71. In this cas,th phtchmical gnratin f1o

2is likl t invlv

ndgnus cllular UVA chrmphrs that hav tt b full charactrizd. Amng RoS, 1o

2is particularl

implicatd in prtin damag in th frm f crsslinksbtwn amin acids72,73. Its significantl lngr half-lif(~110 s in aquus nvirnmnts) givs it a largrsphr f actin than th mr unstabl RoS.

As wll as bing a surc f ractiv xgn, 6-TG itslfis als highl suscptibl t xidatin b RoS. In particu-lar, lw dss f UVA caus a rapid lss f its charactr-istic absrbanc at 340 nm. Tw mar phtprductshav bn idntifid fllwing UVA irradiatin f 6-TGin slutin69,22. Th first f ths, guanin-6-thiguanin(2-amin-6-(2-amin-6-purinl)-thipurin, GSG) isa 6-TG dimr that is frmd in a bimlcular ractinand is unlikl t b a significant DNA phtprduct.oxidatin f th sulphur atm ccurs t gnrat thscnd mar prduct, th highl flurscnt guanin-6-sulphnat (GSo3, FIG. 5). GSo3 is frmd t smxtnt b Tp I phtsnsitizatin and its frmatinis partiall inhibitd b fr radical scavngrs. Tp IIractins invlving 1o

2ar prbabl th mr significant

cntributr, hwvr, and visibl light in th prsncf Rs Bngal a surc f1o

2 fficintl cnvrts

6-TG t GSo3. Analgus ractins ccur in DNA, andDNA GSo3 is frmd whn ithr singl-strandd rdubl-strandd DNA cntaining 6-TG is irradiatdwith UVA r tratd with a mild chmical xidizingagnt74.

Clls cntaining DNA substitutd with a lw lvl f6-TG ar suscptibl t th sam phtchmical rac-tins. Lw UVA dss ar sufficint t prduc a burst fRoS within cllular 6-TG-substitutd DNA and t cn-

vrt a cnsidrabl fractin f th 6-TG t GSo3 (ReF. 69).Gnratin f RoS insid th DNA mlcul itslf isptntiall catastrphic. In additin t xidizing 6-TG tGSo3, RoS can inflict cllatral damag n surrundingnrmal DNA bass75, caus sugar mdificatins, intr-duc DNA strand braks and damag DNA-assciatdprtins (FIG. 5). All f ths ar ptntiall dtrimntalt cllular wllbing. It is likl that RoS damag t cl-lular DNA is rflctd in th snrgistic cttxicit andmutagnicit f 6-TG and UVA69.

GSo3 is a ngativl chargd and bulk DNA adduct. Ithas a cnsidrabl influnc n DNA stabilit and pr-fundl rducs th T

mf duplx ligdxnucltids22.

Hlix dstabilizatin is largl indpndnt f th bas inth ppsing strand, and DNA GSo3 is incapabl f frm-ing stabl pairs with an f th cannical DNA bass. Itis thrfr unsurprising that a tmplat GSo3 is a svrblck t primr xtnsin b DNA plmrass and can

b bpassd nl bY-family DNA polymrass, which hava rlativl rlaxd stringnc76. DNA 6-TG and UVAcmbin with analgus ffct in living clls. Lw dssf UVA caus rapid inhibitin f DNA rplicatin inclls with DNA 6-TG. This triggrs mnubiquitlatinf th prlifrating cll nuclar antign (PCNA) rpli-catin factr gnrall rgardd as a signal fr thrcruitmnt f spcializd DNA plmrass t bpassrplicatin-blcking DNA lsins. Th sam tratmntsals induc thp53-dpndnt DNA damag rspns22.Stabilizatin f th p53 prtin and inductin f p21 (alsknwn as CDKN1A) ccur with th xpctd kintics,but at highr UVA dss th transcriptinal rspns f

Figure 4 | absob st o 6-TG d G. Canonical DNA purines, for example,

guanine (shown here), have no significant absorption of ultraviolet radiation at

wavelengths longer than 300 nm. Replacement of the 6-O atom by sulphur (6-TG see

chemical structure) causes the absorbance maximum to shift into the UVA region

(~340 nm).

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9/13

ROS

RadicalOH hydroxylO2

superoxideO2R peroxylOR alkoxylNO nitric oxide

Double-strand/single-strand breaks

N

NN

N

H2N

S

dGSO3

O

OO

dR

HN

N

O

O

dR

OH

OH

CH

Thymidine glycol

HN

N N

HN

O

H2N

O

dR

Oxo8deoxyguanosine

Possible detrimental effects of intracellular ROS

DNA damage

Oxidized bases

Lipid peroxidation

Protein damage (1O2)

Aldehydes

Amino-acidcrosslink

Damage to DNA-associated proteins

DNA adducts

Non-radical1O2 singlet oxygenONOO peroxynitriteH2O2 hydrogen peroxide

3

Y-family DNA polymerases

Y-family DNA polymrass

hav a mor opn activ sit

than A- and B-family DNA

polymrass and can

accommodat covalntly

modifid tmplat DNA bass.

This allows thm to insrt

nuclotids opposit damagd

bass. This bypass mod

allows th rplication of

damagd DNA to continu,

although oftn at a cost to

fidlity.

p21 is attnuatd, suggsting dirct inhibitr ffcts fDNA damag n transcriptin. Th cmbind ffctsf 6-TG and UVA n rplicatin and transcriptin arcnsistnt with th intrductin f bulk DNA adductsand prbabl rflct th frmatin f DNA GSo3.

DNA is nt th nl targt f phtchmicall gnr-atd RoS. Phtchmical prtin damag principallb1o

2, which is frmd in significant amunts during th

phtchmical dstructin f 6-TG is a cntributr tsvral pathlgical cnditins including lns dgnra-tin in th and altrd drmal cllagn functin77,78.Crsslinkd r aggrgatd prtins ar mar prducts fintracllular 1o

2, which can ract with armatic (phnl-

alanin, trsin and trptphan), basic (arginin, lsinand histidin) and sulphur-cntaining (mthinin andcstin) amin acids. Amng ths, histidin is th marcasualt with th highst rat cnstant fr ractin with1o

2and histidin appars t b ssntial fr 1o

2-mdiatd

prtin crsslinking79. Th gnratin f1o2

frm DNA6-TG and UVA crats what might b a uniqu dangr frprtins intimatl assciatd with DNA. Althugh 1o

2

mlculs hav a rlativl lng half-lif (f th rdr f

micrscnds rathr than th nanscnds f thr RoS),th ract cls t thir sit f frmatin. Th multiprtinrplicatin and transcriptin cmplxs as wll as his-tns and nn-histn DNA binding prtins ar allptntial targts fr xidatin. PCNA is n casualt fDNA-gnratd 1o

2. This imprtant DNA rplicatin

and rpair factr is a hmtrimric cmplx that actsas a sliding clamp t tthr DNA plmrass t DNA,thrb imprving thir prcssivit and accurac. AftrUVA tratmnt f clls cntaining DNA 6-TG, thsubunits f PCNA ngagd in rplicating thipurin-substitutd DNA bcm cvalntl bund t nanthr74. Th prcis structur f th PCNA cmplx

and th bilgical cnsquncs f its frmatin hav tt b dtrmind. Cvalnt linkag btwn th subunitsf th PCNA trimr is likl t cmprmis its functin,hwvr, bcaus lading f PCNA nt DNA rquirstransint pning f th trimr. In additin, thr is nrasn t supps that PCNA is th nl xidizd prtinand its cvalnt mdificatin illustrats th vulnrabilitf DNA-assciatd prtins in clls that ar xpsd t6-TG and UVA.

RoS-mdiatd DNA damag is a mar surc fmutatin. Clls ar adaptd t dal with th thrat frmth RoS that ar invitabl gnratd during nrmalarbic mtablism. Cnditins that significantl xcdths lvls saturat cllular dfncs and induc xida-tiv strss. Th phtchmical ractins f DNA 6-TGar ptntiall hazardus fr tw rasns. First, althughhigh lvls f UVA aln gnrat RoS, DNA 6-TG isa significant UVA snsitizr and ma rduc th UVAds that is rquird t caus damag t within th rangf nrmal sunlight xpsur. Scnd, th RoS ar gnr-atd in DNA itslf. As ths ar likl t ract cls tthir sit f frmatin, DNA, and assciatd prtins

rspnsibl fr its accurat rplicatin, ar frnt-lintargts. A plausibl utcm f th phtchmical rac-tins f DNA 6-TG is an incrasd burdn f mutatinand an incrasd risk f malignanc.

Clinical implications therapy-related cancer

Azathiprin is dsignatd as a human carcingn largln th basis f pidmilgical vidnc. Althughunquivcal, th data prvid n indicatin as t thmchanism b which cancrs might aris. In th fl-lwing sctins w cnsidr hw th prprtis f aza-thiprin, in particular f DNA 6-TG, might influncth dvlpmnt f particular malignancis.

Figure 5 | rti oxyg sis (rOS) d otti dmg to u momous. Radical and non-

radical forms of ROS can cause direct damage to DNA constituents in the form of oxidized bases and deoxyribose, and

cleavage of the phosphodiester backbone to form single- or double-strand breaks. They can also initiate reactions

that generate secondary reactive species, such as aldehydes, that can also form DNA lesions. DNA 6-thioguanine isa particular target for oxidation and one of the major products (GSO3) is shown. Proteins are also vulnerable to ROS,

particularly to 1O2

which reacts with aromatic, basic and sulphur-containing amino acids.

R E V I E W S



10/13

Ulcerative colitis

A form of inflammatory bowl

disas in which inflammation

affcts th larg intstin or

colon and consists of

charactristic ulcrs or opn

sors.

Crohns disease

A typ of inflammatory bowl

disas. It is charactrizd by

inflammation across th ntir

wall of th affctd mucosa

and can affct any part of th

gastrointstinal tract.

Acute myeloid leukaemia. Mlsupprssin is ds-limiting fr thipurin tratmnt and dss ar adustdt th maximal lvl cmpatibl with maintaining satis-factr bld cll cunts. Thipurin tratmnt placs thparticularl snsitiv bn marrw clls undr a slctivprssur that mimics labratr xprimnts dsigndt slct rar MMR-dfctiv clls29,48,49. Thrap-rlatdAML is a ptntial cmplicatin f thipurin thrap fracut lmphblastic lukamia80,81 and is als vrrpr-sntd amng rgan transplant patints. Th rlativ riskis 5.5 fr hart and lung transplants and 2.1 fr cadavrkidn rcipints49. Unlik th cancrs that ar knwn tinvlv viruss82, AML is lat in nst and th incidncnl divrgs significantl frm that f a matchd immu-ncmptnt ppulatin aftr a lag f ~35 ars49. Thazathiprin-ds dpndnc in bth transplant grupssuggsts a causal cntributin frm th thipurin. Itis particularl ntwrth that th frqunc f micr-satllit instabilit (MSI) is high amng transplant-rlatdAML cass. MSI is gnrall rathr infrqunt (


11/13

rsults in ilclic inflammatin, incrasd tumursand incrasd frquncis f xidativ strss-rlatdsignatur mutatins97.

Th gastrintstinal mucsa in IBD is charactrizdb th prsnc f activatd immun clls. Phagcticclls, principall nutrphils and macrphags, prducRoS and thr mtablits that caus pathlgical tissudstructin. Prlifrating clnic pithlial clls withinthis intns inflammatr micrnvirnmnt ar liklt b suscptibl t th mutagnic ffcts f chrnicallgnratd RoS. In rdr t gnrat ptntiall harmfulDNA xidatin prducts phagct-drivd RoS mustb abl t diffus acrss cllular mmbrans. H

2o

2sms

th mst likl ffctr mlcul as it can gnrat DNAdamaging oH radicals b th Fntn ractin. DNAdamag b th prducts f lipid prxidatin is alspssibl.

In viw f th particular suscptibilit f DNA6-TG t xidatin and th dmnstratd mutagnicitf cmbind DNA 6-TG and RoS, it sms pssibl thatth miliu f xidativ strss ma rprsnt a particular

mutatinal hazard fr DNA 6-TG-cntaining clnicpithlial clls f patints tratd with azathiprin.

Althugh ths cncrns still rquir validatin bdtaild clinical studis, it sms rasnabl t nsurthat patints tratd with thipurins fr IBD rcivlng-trm survillanc fr pssibl clrctal cancr.

Concluding remarks

ovr th past fiv dcads cuntlss patints, man suf-fring frm chrnic dbilitating disass, hav bnfitdfrm tratmnt with thipurins, and th us f azathi-prin as an immunsupprssant was instrumntal in mak-ing rgan transplantatin frm unrlatd dnrs a ralit.In th mdrn ra f pharmacthrap this lngvit

f us is a tstimn t thir succss. Fllwing a mrdtaild undrstanding f thipurin mtablism and thdvlpmnt f pharmacgntic tsting it is anticipatdthat thir saft prfil, particularl with rspct t m-ltxicit, will cntinu t imprv. It is undisputd thatthipurin tratmnt is assciatd with cancr. This isclarl stablishd fr hamatlgical and virus-assciatdmalignancis, and skin cancrs. W hav dscribd hwth prsnc f th thibas altrs th chmistr f DNAand hw this might facilitat th dvlpmnt f can-cr. on pstulatd rut invlvs th incrasd rat fspntanus mutatin that accmpanis inactivatinf th MMR sstm. An altrnativ rut invks xi-datin and xidativ strss, a widsprad cnditin thatis particularl prvalnt in inflammatr disrdrs. Wwuld mphasiz, hwvr, that th individual patintrisk is likl t b small, and in man cass is far ut-wighd b th bnfits f tratmnt. This is bvius inth cas f lif-saving rgan transplantatin. In th casf chrnic nn-lif-thratning cnditins such as rhu-matid arthritis, th quatin is prhaps lss clar-cut. In

an vnt, th ptntial cmplicatin f thrap-rlatdcancr shuld nt b ignrd.

Th past fw ars hav sn an xplsin in thdvlpmnt f nvl immunsupprssants, man fwhich ar rplacing sm f th mr stablishd trat-mnts such as azathiprin and ciclsprin (FIG. 1). Thmds f actin f ths nwr drugs, insfar as thar knwn in dtail, diffr significantl frm that f ththipurins. Th hav nt bn in clinical us fr lngand th implicatins fr cancr dvlpmnt canntb knwn at this arl stag. In viw f th lng-trmnatur f immunsupprssin, cntinud awarnss fthrap-rlatd cancr as a pssibl dlad sid ffctf tratmnt sms prudnt.

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102.Anstev, A. V., Wakelin, S. & Reynolds, N. J.

Guidelines for prescribing azathioprine in

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AcknowledgementsWe are indebted to the numerous laboratory colleagues,

past and present, who helped shape many of these opinions,

especially to J. Offman, P. ODonovan and C. Perrett, who

contributed particularly to our studies of therapy-related

cancer. Many of the ideas expressed in this article have

been developed and refined over several years. We thank M.

Bignami, Y.-Z. Xu, P. Swann, G. Opelz, C. Harwood and J.

McGregor for their significant contributions to this.

DATABASESEntrez Gene:http://www.ncbi.nlm.nih.gov/entrez/query.

fcgi?db=gene

ATR|ATRIP | CDKN1A | CHEK1 | HPRT1 | IMPDH|MLH1|

MSH2|MSH6 | p53 | PCNA | PMS2 |PPAT|TPMT

FURTHER INFORMATIONPeter Karrans homepage:http://science.cancerresearchuk.

org/research/loc/london/lifch/karranp/

all lInkS are acTIve In The OnlIne pDf

R E V I E W S
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genehttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genehttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=545http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=545http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=84126http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=84126http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=1026http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=1111http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=3251http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=3614http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=3614http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=4292http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=4292http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=4292http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=4436http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=4436http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2956http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2956http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=7157http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=5111http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=5395http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=5471http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=5471http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=5471http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=7172http://science.cancerresearchuk.org/research/loc/london/lifch/karranp/http://science.cancerresearchuk.org/research/loc/london/lifch/karranp/http://science.cancerresearchuk.org/research/loc/london/lifch/karranp/http://science.cancerresearchuk.org/research/loc/london/lifch/karranp/http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=7172http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=5471http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=5395http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=5111http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=7157http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2956http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=4436http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=4292http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=3614http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=3251http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=1111http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=1026http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=84126http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=545http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genehttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene

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