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1991;51:5430-5432.Cancer ResDonald C. Malins and Russom Haimanotof the Female BreastMajor Alterations in the Nucleotide Structure of DNA in Cancer

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[ CANCER RESEARCH 5 !, 5 43 0- 54 32 . O cto be r I , 1 99 1]A dvances in B riefMajor A lterations in the N ucleotide Structure of DNA inC ancer of the Fem ale B reast1D onald C . M alins and R ussom H aim anotEnvi ro nme nt al B io ch em is tr y P ro gr am , Pa ci fi c N or thw es t R es ea rc h Founda ti on , S ea tt le , Wa sh in gt on 98122

AbstractD NA of invasive ductal carcinom as from five wom en w as analyzed for

s tr uc tu ra l a lt er at io ns in t he pur in e nuc le ot id es u si ng g as c hr omat og ra phy-n iass sp ectrom etry w ith selecte d ion m on itorin g. T he resu lts w ere compared to those for a normal DNA control. The carcinoma DNA showedd rama tic ally h ig he r c on ce ntr atio ns o f t he b ase m od ific atio ns 8 -h yd ro xy -g ua ni ne , 2 ,6 -d iami no -4 -h yd ro xy -S -f ormam idopyr im id in e, a nd 8 -h yd ro x-yad en in e. F or exam ple, th e con cen tr atio n of total id en tified b ase m od ifications represented a m ore than 9-fold increase over the control value.B ase m od ific atio ns o f th is ty pe , w hic h a ris e fr om r ad ic al- in du ce d h yd ro x-ylation an d cleavage r eaction s of th e p urin e rin g, lik ely p lay a m ajor rolein initiation and probably contribute to the further transform ation ofn eop lastic c ells in c an cer of th e fem ale b reast.

IntroductionThere is abundant evidence suggesting that the hydroxylradical ('O H) p ro duce s alteration s in th e stru ctural integrity ofthe DNA bases (1-3). In vivo, unless the modified DNA ispromptly repaired, m iscoding may occur in replication (4)which m ay result in the form ation of neoplastic cells (1,2, 4).In this regard, a study w ith E scherichia coli (4) indicated thatthe 8-hydroxydeoxyguanosine residue, w hich arises from theattack of the -O H on the purine ring (5), has an overwhelm ingeffect on tem plate-directed DNA synthesis in that it causesm isreplicatio n at its o wn po sitio n as w ell as at neigh bo ring b asepositions. T hus, there is persuasive evidence for the conceptthat the introduction of oxygen into nucleotide structure is anin itiatin g step in m utage nesis an d carcino gen esis (2 , 4 ).W e have show n, using the E nglish sole (P arophrys vetulus)carcinogenesis m odel (6), that the -O H-induced base m odifications 8-O H-Gua,2 Fapy-G , 8-OH -A de, and Fapy-A (Fig. 1)

c an b e s tru ctu ra lly a nd q ua ntita tiv ely e lu cid ate d in tis su es u sin gGC-M S/SIM (7-9). Studies with this vertebrate model alsodem onstrated that substantial elevations in 8-O H-G ua, Fapy-G , 8-OH-Ade, and Fapy-A occurred in the D NA from hepaticcarcinom a tissue com pared to baseline concentrations in norm al tissue (7-9). Thus, it was suggested that the DN A m odificatio ns (w hich h ad n ot prev io usly b een id en tified in an y an im alsystem ) likely played a causative role in the form ation of thec ar cinoma s ( 7- 9) .A recent study is notew orthy w ith respect to the role playedby the -OH in modifying DNA. It was shown in vitro (10) thatthe *OH reacts with calf thymus DNA to yield a variety ofnucleotide base derivatives such as 8-OH -Gua and Fapy-G .Rece iv ed 7 /1 7/ 91 ; a c cept ed 8 /1 6/ 91 .T he c osts o f p ub lic atio n o f th is a rtic le w ere d ef ra ye d in p art b y th e p ayme nto f p ag e c ha rg es. T his a rtic le m ust th er efo re b e h ere by m ark ed a dv ertise me nt ina cc or da nc e w it h 1 8 U .S .C . S ec tio n 1 73 4 s ole ly t o in dic at e t his f ac t.' This work w as supported by United States A rm y M edical R esearch andDeve lo pment Command Gran t DAMD17 -88-Z-8043.2 The abbreviations used are: 8-OH -G ua, 8-hydroxyguanine; Fapy-G ,2 ,6-d iamino-4-hydroxy-5 - fo rmamidopyrimid ine ; 8 -OH-Ade, 8 -hydroxyadenine ;Fapy -A , 4 ,6 -d iam ino-S -f ormamidopyr im i di ne ; GC-MS/S IM, g a s chroma to gr a-p hy-mas s s pec tr ome tr y w it h s el ec ted i on moni to ri ng ; TMS , t rimet hy ls il yl .

T his reaction w as m ediated by the iron ion-dependent super-o xid e rad ical-g en eratin g system h yp ox anthine /x anthine o xidase. The reaction produced, for example, 8.80 nmol Fapy-G /m g DN A. Inhibition of product form ation occurred w ithm an nito l, d im eth yl su lfo xid e, S up ero xid e d ismuta se , a nd c ata -lase, thus indicating that -OH was formed from H2O2 by anO2~ -a ss iste d F en to n re ac tio n. L ow c on ce ntra tio ns o f b ase modifications in norm al DNA , such as from calf thym us (10) andE nglish sole (7-9), are readily identified at or near thresholddetection lim its using the G C-M S/SIM technique. In norm alcells, a prim ary defense against 'OH -induced elevations inDN A base m odifications is provided by the glycosylases andother enzym es that participate in the excision repair process,as well as by antioxidants such as glutathione (11-13). However, excessive concentrations of the -O H overcom e norm aldefense m echanism s and leave the D NA at risk from oxidativem odifications (13) that are believed to be causally related toc arc in og en esis (2 ). T he p ro cess of H -induced D NA m odification is reflected in the present findings on invasive ductalc arc in om a o f the fem ale b re ast.M aterials and M ethodsE xcised carcinom a tissue from five fem ale patients w as show n m ic ro sc op ic al ly t o c on ta in i nv as iv e d uc ta l c ar ci nom as ; h ow ev er , e xamin atio n o f th e ex cise d su rgica l m argin tis su e rev ea led no ev id en ce fo rneoplasia, although there w as som e evidence for other m icroscopicc ha ng es ( e.g ., f ib ro cy ti c) . R es id ua l c ar ci noma -c on ta in in g a nd e xc is eds ur gi ca l m ar gin t is su e w as p la ce d i n l iq uid n it ro ge n immed ia te ly a ft errem oval and m aintained at -70 C prior to extraction of the D NA ,

w hich w as u nd erta ken a s d escrib ed p rev io us ly (1 4). DNA w as h yd ro -ly ze d an d TMS d eriva tiv es w ere p re pa red u nde r a n atm osp here o f p uren itr og en ( 15 , 1 6) . T h e TMS d eri va tiv es w er e a na ly ze d b y GC -MS /S IM(7-9, 15, 16) using a H ew lett-P ackard M odel 5890 m icroprocessor-c on tr ol le d g as Chr om at og ra ph i nte rfa ce d to a H ew le tt- Pa ck ar d Mo de l5970B m ass selective detector. T he injector port and interface w ereboth m aintained at 250 C .T he colum n w as a fused silica capillarycolum n (15.0 m ; 0.2 mm inner diam eter) coated w ith cross-linked 5%p he ny lm eth yl sil ic on e g um p ha se ( fi lm t hic kn es s, 0 .3 3 ^m ). T he c olumnte mp era tu re w as inc rea sed from 1 20 to 1 76 Ca t 3 C/m in a nd fro m1 76 to 2 50Ca t 6 /m in ,a fte r i nit ia lly b ei ng h eld f or 1 .5 m in a t 1 20C.H elium w as used as the carrier gas w ith a linear velocity of 23.5 cm /sth ro ug h th e co lu mn (15 , 16 ). T he am ou nt o f TMS h yd ro ly sa te in je ctedonto the column was about 0.7 tg-Q uantitation of the m odifiedn uc le ot id e d er iv at iv es w as u nd ert ak en o n th e b as is o f t he p ri nc ip al i on s,such as m /z 442 for the TMS derivative of F apy-G (15, 16).Fapy-A , 2 ,4 ,5 -t ri am ino -6 -h yd ro xypy rim id in e s ul fa te , a nd 8 -b romo-a den ine w ere p urc hase d from S igma C hem ic al C o. an d 8 -OH-G ua w aso btain ed fro m th e C hem ic al D yn am ics C orp . T he 8 -OH-A de a nd F ap y-G wer e s yn th es iz ed in o ur l ab or at or ie s f rom 8 -b romo ad en in e a nd 2 ,4 ,5 -t ri am ino -6 -h yd ro xypy rim id in e s ul fa te , r es pe ct iv el y, a nd pur if ie d b y r e-c ry s ta ll iz a tion (17) .

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DNA BA SE M O DI FI CAT IO NS I N BR E AST C ANC ERNH,

k NVO H

8-OH-Ade

NH,

H,Kapy-A

UNO

OH Hr(I

HH,8-OH-Gua Fapy-G

F ig. 1. M odifications arising from the attack o f the -O H on C 8 of the purinering of adc nine and guanine . A s an exam ple, the am ine at position 6 o f adcnineprom otes an e lectron de fic iency al C -8 w hich results in the attack of the -O H,s ub se qu en t c le av ag e o f th e p ur in e r in g, a nd f orma tio n o f t he f ormami do py rimi -d in e s tr uc tu re ( 5. 8 ).R esu lt s a nd D iscu ssion

T he ca use of b reast can cer is e ssentially u nkn ow n; h ow ev er,c an ce r fo rm atio n is lik ely to in vo lv e s tru ctu ra l m od ific atio ns inth e n uc le otid e b ase s th at a ffe ct temp la te -d ire cte d DNA sy nth esis (4 ). T hus, it is sig nifican t th at th e p resen t fin din gs rev ealeddram atic d ifferen ces in th e co ncentratio ns of 8 -OH-G ua, F ap y-G , an d 8 -OH-A de w ith resp ect to th e co ntrol an d th e carcin om atissues (Fig. 2). T he values for 8-O H-G ua, Fapy-G , and 8-O H-Ade in the control were 0.13 0.03 (SD), 0.08 0.08, and0.22 0.05 nm ol/m g, respectively (Fig. 2). The respectivev alues fo r th e carcin om a tissue s w ere 8 - to 17 -fo ld hig her (1 .2 6 0.78, 1.33 0.97, and 1.67 1.86 nm ol/m g D NA ). In boththe co ntro l an d th e carcinoma tissu es, F apy -A w as p resen t o nlyat low levels near the lim its of detection of the GC-M S/SIMtech niqu e (0.04 nm ol/m g DNA ) (F ig. 2 ). A cco rd in gly , F ap y-Ais not a prominent indicator of altered DNA in breast cancerin contrast to the other base modifications. There was not asignificant difference between the calf thym us and surgicalmargin DNA with respect to any of the base modifications;how ever, a significant difference did exist betw een the D NAfrom th e su rg ic al m arg in an d th e carcin om a tissue w ith resp ectto 8-OH-Gua (P < 0.01), Fapy-G (P < 0.03), and 8-OH-Ade(P < 0.05). On a m atched pair basis (surgical m argin versuscarcinom a), the concentrations of each of the above base m odification s w ere su bstan tially hig her in th e carcinoma, w ith theex cep tio n o f FBT -5 w hich h ad relativ ely lo w co ncen tratio ns o fth e b ase lesio ns (see F ig . 2 legen d).In stu dies w ith th e E ng lish so le carcin og en esis m od el (7 -9 ),w e fou nd th at th e relative ly lo w co nc entra tio ns o f b ase m od ification s in n orm al tissu es w ere w ith in a relativ ely narro w rang e,close to the threshold of detection of the G C/M S-SIM m ethod.In this regard, the present values with calf thym us DN A wereno t ap preciab ly d ifferent from tho se o btain ed b y o urselv es (7-9) and other workers (10, 15, 16). Moreover, in an initiala ttemp t to u nd ersta nd b as e le ve l c on ce ntra tio ns o f th e mod ifie dn uc le otid e d eriv ativ es in h um an tiss ue s, w e s tu die d le uk oc yte sfrom th e b lo od o f tw o a pp are ntly n orm al in div id ua ls . T he v alu esobtained w ere consistently low : 0.20 and 0.23; 0.12 and 0.14;0.01 and 0.07; and 0.04 and 0.04 nmol/mg DNA for 8-OH-G ua, 8-O H-A de, Fapy-G , and Fapy-A , respectively/' T here isevidence to suggest that surgical margin tissue may not bem icroscopically norm al (18). N evertheless, as indicated, interms of the DNA bases examined, the surgical margin DNAwas not significantly different from the calf thymus DNA.A cc ord in gly , th e c alf th ymus d ata w hic h h av e p re vio us ly s erv ed

' D . C . M alin s. R . H aim anot . a n d M . Bean , un pub lished r esu lts.

as a standard for "normal DNA" (10, 15, 16) are compared tothe carcinom a data in Fig. 2.O ve ra ll, th e p re se nt fin din gs p ro vid e p ers ua siv e e vid en ce fo rsu bstan tial -OH-in duce d a lteratio ns h av in g tak en p lace in thepurine nucleotides of D NA from the breast carcinom a. M oreover, it seems unlikely that the radical attack on the DN A wasessentially confined to 8-O H-G ua, F apy-G , and 8-O H-A de. Itis p ro bable, fo r ex am ple, th at th e py rim id in e nu cleo tide s w erealso m odified, although thus far w e have not exam ined thesemod ific atio ns in s uffic ie nt d eta il to e lu cid ate th eir re le va nc e toc ar cinogenes is . A ccor di ng ly , th e s ub sta ntia l b as e modi fi ca tio nsreported are likely to reflect only a partial assessm ent of theo xid ativ e ch an ges in flicted o n th e breast DNA .To our knowledge, the present study is the first to exam ineD NA base m odifications in any m am malian tissue on a structural and quantitative basis and, as such, the findings provide au niq ue o pp ortu nity to ev alu ate th eir sig nifican ce in relatio n toth e patho bio lo gy o f b reast can cer. In this resp ect, th e p rese nceof the relatively high concentration of 8-O H-Gua in the D NAof the carcinom a tissues seem s especially relevant in view ofth e e vid en ce d emon stra tin g th at 8 -h yd ro xy de ox yg ua no sin e h asan o verw helm in g effect in cau sin g m isrep licatio n in tem plate-d irecte d DNA sy nth esis (4). T he sign ifica nce o f th e o th er DNAm odifications in this regard can be ascertained only from further studies. Considering the special need for m aintaining the

aa>

i= 300^

JLF ig . 2 . DNA b ase m od if ic atio ns (n mo l/m g DNA ) in fiv e f em ale b re as t tu mo rs(FB T) of the invasive ductal carcinom a type are com pared to those of a (calft hymu s) c on tr ol. T he DNA b as e mo dif ic at io n c on ce nt ra tio ns r ep re se nt a n a ve ra geo f d up li ca te a na ly se s b y GC -MS /S IM . T he re w er e s ig ni fi ca nt d if fe re nc es amon gth e c on ce ntr atio ns o f 8 -OH-Gu a ( P -i 0 .0 1), F ap y- G ( P < 0 .0 2) . a nd 8 -OH-A de

(P < 0 .0 5) w ith res pect to th e ca rcin om a tiss ue a nd th e c on tro l (five re plica tea na ly se s); h ow ev er, th e n uc le otid e b ase p ro file o f F BT -5 w as n ot sig nif ic an tlydifferent, a lthough the value for 8-O H-G ua w as tw ice that of the control. T hereason for this anom aly is not readily apparent, although it m ay b e related to ap au c it y o f n eopl as ti c c el ls .5431

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D NA B AS E MOD IF IC AT IO NS IN B RE AS T CA NC ERstru ctu ra l in teg rity o f DNA th rou gh en zymatic an d o th er p ro cesses (11, 12), the substantial H -in duc ed m od ificatio ns inth is m olecu le are likely to be cau sally related to th e ne op lastictransform ations in the breast. H ow ever, the origin of the -O Hth at po ten tially in itia tes th e b ase m od ification s is un clea r, although one possibility is that this radical arises from H2O2g en erated th ro ug h th e cy to ch rom e P -4 50 -m ed iated ox id ationo f e stro ge n (1 9).A sa lie nt fe atu re o f tumo r c ells is th eir c on stitu tiv e p ro pe ns ityto generate high concentrations of H2O2 (20). This suggeststhat the -OH attack on DNA in the carcinoma itself may beasso ciated w ith escalation s in H 2O 2 c on cen tration s and sub sequent formation of the -OH. That is to say, at some stage intumor d ev elo pm ent, transfo rm ed cells b ecome in effect "auto-muta ge nic ," b rin gin g a bo ut p ro gre ssiv e a lte ra tio ns in th e DNAas a con seq uen ce o f co nstitu tive H 2O 2 g eneratio n. M oreo ver,o n the b asis o f th e p resen t resu lts, th e "an tio xid an t" cap ab ilityattributed to tum or cells (21) m ay be accounted for, at least inpart, by trapping of the -O H radical through its reactions w ithDNA. For example, in sample FBT-1 (Fig. 2) the combinedconcentrations of 8-O H-G ua and 8-O H-A de alone are equivalent to 7.7 nm ol of H2O2 or -OH, assum ing a 1:1 m ol ratio (5).In add itio n, th e resistan ce to lysis asso ciated w ith n eo plasticcells (22) m ay also reflect to som e degree the presently demonstrated tendency for DNA to act as a trapping agent for theOH,hich h as w ell k now n cy tolytic p ro perties (2 3).Studies of H 2O 2 generation in tum or cells (20) suggest thatth e re la tiv ely h ig h c on ce ntra tio ns o f th is o xid an t le ad to g en eticinstability (20, 24, 25). In this regard, the present findingssuggest that the D NA base structure in the breast carcinom a isp rog ressiv ely m od ified by th e 'OH a nd is th us likely to p ro du cea variety of genetically altered cell types, to possibly includethose capable of m etastasis. Overall, it seem s likely that theO H-inducedase m odifications of DN A found in fem alebreast tum ors w ill also be m anifested by other types of cancer.In addition, the sensitive G C/M S-SIM m ethodology shouldp ro ve to be v alu ab le as a d iag nostic too l for p red icting , th rou ghD NA base m odifications, the occurrence of cancer in a varietyo f tiss ue s (9 ).AcknowledgmentsApp re cia ti on i s e x pr es se d t o The L ab ora to ry o f P ath ol og y o f S ea tt le ,Inc., for breast tissue and to D rs. W illiam B . H utchinson, M ichaelB ean , E ric H olm es, an d Jo hn H ou ck for co nsu lta tio n.

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