p53 tumor suppressor gene mutation in early esophageal

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1994;54:4342-4346.Cancer ResHongkun Gao, Li-Dong Wang, Qi Zhou, et al.Populations in Henan, ChinaPrecancerous Lesions and Carcinoma among High-Risk

Tumor Suppressor Gene Mutation in Early Esophagealp53

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[C AN CE R R ES EA RC H 5 4. 4 34 2-4 34 6, A ug us t 1 5. 1 " 4 |

p53 Tumor Suppressor Gene Mutation in Early E sophageal Precancerous Lesionsand Carcinoma among H igh-R isk Populations in H enan, China1Hongkun Gao, Li-Dong W ang, Qi Zhou, Jun-Yan Hong, Tong-Yuh Huang, and Chung S. Yang2l.a bo ra to r\ fo r C an cer R esea rc h, C olle ge o f I'lia rm ac v, R utg ers U niv ersity, P isc ata wa y, N ew Je rsey 0 88 55 H .G ., L -D . W ., Q . Z ., T -Y . H ., J- Y. H ., C . S . Y .j, a nd H er a nIn stitu teo f M ed ic al S cie nce s, H en an M ed ica l U niv ersity , Z he ng zh ou 4 50 05 2, C hin a /L -D . W ., Q . Z .

ABSTRACTTo understand whether /o.i gene mutation is an early or late event in

e so ph ag ea l c ar cin og en esis , b io ps y s am ple s o f e so ph ag ea l e pit he lium fr omsym ptom -fr ee sub jects in a h igh in cid en ce area, H uixian cou nty of H en anProvince, China, were analyzed. M utations in exons 5, 6, 7, and 8 of p53w er e a na ly ze d b y sin gle -s tr an d c on fo rm atio n p olymo rp hism a na ly sis a ndD NA sequencing. A mong the 37 biopsy sam ples show ing accum ulation ofp 53 p rotein in immu noh istoch em ical stain in g, m issen se m utation s of p 53gene w ere delected in 1 o f 3 sam ples w ith norm al epithelia, 3 of 23 sam pleswith basal cell hyperplasia, and 4 of 11 samples with dysplasia. Allmutations occurred at exon 5 with 3 at codon 175, 2 at codon 176, and 1each at codons 159, 135, and codon 132. O f the 8 m utations, there were 3G to A tran sition s an d 3 G to T tran sversion s. T o u nd erstan d th e m utationsp ectr um an d p ossib le cau sative fac tors of esop hageal can ce r in th is area,su rgic ally re sected h um an p rim ary esop hageal carcin om as from L in xiancounty were analyzed for p53 gene mutations in exons 5, 6, 7, and 8.Mutations were detected in 16 of 29 samples (55% ). Twelve samplescon tain ed d ifferen t m issen se p oin t m utation s, w ith 75% tran sition s (7 G toA and 2 A to G) and 25% transversions (2 G to T and l G to C). Most ofthe mutations were located at either exon 5 or exon 7. A deletion and anin sertion of n ucleotid es lead in g to fram e-sh ift m utation s w er e d etected ineach of two other samples. The results demonstrate that p53 proteinaccum ulation and gene m utation m ay occur at very early stages of esophage al carcino gen esis. In c arcin om as, th ere w as a h igh er freq uen cy ol'/o.fgene mutations, which accounts for m ost of the cases with p53 proteinaccumulation.

INTRODUCTIONE sophugcal cancer is one of the m ost common cancers w orldw idew ith s ha rp v ariatio n in its in cid en ce rates a mo ng d iffe re nt co un tries,d is tin ct g eo grap hic a rea s, a nd d iffe re nt eth nic g rou ps (1 ). In W este rncountries, heavy cigarette sm oking and alcohol intake are m ajor riskfactors, w hereas in T hird W orld countries, exposure to dietary carcinogens and nutritional deficiencies are believed to be m ajor etio-logical factors (1-5). Linxian and the nearby counties in the H enan

P rovince in northern C hina have received w orldw ide attention overthe past decades because of their high esophageal cancer incidence(2). Ingestion of dietary carcinogens, in particular A '-nitroso compounds and their precursors, has been im plicated (2, 6, 7). E levatedlevel of O ''-m ethyldeoxyguanosine in the esophageal cancer tissueshas been reported (8). A '-N itroso com pounds induced esophagealtumors in rats and m ice (9-11) and may be causative factors forhum an esophugeal cancer. H ow ever, the identities of the causativefactors of hum an esophageal cancer still rem ain to be studied.M an y p ath olo gic al a nd ep id em io log ica l stud ies su gg es t th at m alignant transform ation of hum an esophageal m ucosa is a progressivep ro ce ss (2, 1 2-1 6). O ur p re vio us stu dies rev ealed th e a cc um ula tio n o fRec ei ve d 3 /3 1/ 94 ; a cc ep te d 6 /1 3/ 94 .T he costs of publication of this article w ere defrayed in p art by Ihe pay ment o f pagec har ges . T his artic le m us i th eref ore b e h er eb y m ar ked ad ve rtis em en t in a cco rd an ce w ith1 8 U .S .C . S ec tio n 1 73 4 s olely to in dic ate th is fa ct.1 This w ork w as supported by N IH G rant C A37 037 and N ational Institute of E nv i

r onme nt al H ea lt h S ci en ce s C en te r G ra nt E S0 50 22 .o whom requests for reprints should he addressed, at Laboratory for CancerR es ea rc h. C ol le ge o f P ha rm ac y. R ut ge rs U ni ve rs it y. P is ca ta wa y, N J 0 88 55 .

p 53 p ro te in in es op ha ge al ep ith elia w ith d ysp la sia an d hy perp lasia a ndeven in the papillae of near norm al epithelia of biopsy sam ples fromindividuals w ithout cancer (17). T he current study extended this lineo f inv estig ation a nd an aly ze d p 53 m utatio ns in p rec an ce ro us les io nso f t he e so ph ag us .M utations of the p53 tum or suppressor gene have been identified inm any types of hum an cancers (18-21). M ore than 300 different pointm utations have been reported and several hot spots have been locatedin the p53 gene, such as at codons 175, 248, and 273 (22). One hotspot of p53 m utation at codon 249 was detected in hepatocellularcarcinom as in patients from Q idong, C hina, and S outh A frica w hereaflatoxin B, w as a common risk factor (23, 24). In the same type ofcancer from other areas w here the populations w ere not exposed toaflatoxin B ,, m utations w ere detected at diverse positions of the p53gene (25, 26). T his suggests that the m utation pattern of p53 could berelated to the nature of the exposed environm ental carcinogens (21).It is possible that the high incidence of esophageal cancer in H enan,C hina, is due to particular environm ental carcinogens: and that m utation hot spots of the p53 gene exist in esophageal carcinom as andp re can cero us le sion s. T o e xam in e th is p ossibility , th e m uta tio n s pe ctrum of the esophageal carcinom as w as analyzed and com pared w ithth at o f th e p re ca nce ro us lesio ns .

MATERIALS AND METHODSS pe cim en C olle ctin g. T he b io psy s am ple s w er e c olle ct ed f rom s ymptom-

free subjects from H uixian, C hina. T he surg ically resected spec im ens w ereo bta in ed fro m p atie nts w ith p rim ary e so ph ug ea l c arc in om a in L in xia n. C hin a.T he patien ts had not receive d any radiation thera py or chem otherapy beforesurgery. The pathological diagnosis was based on hematoxylin and eosinstaining a nd a ll sam ples w ere invasive S CC .'I mmun oh isto ch em ic al A na ly sis o f p 53 P ro te in . Immun oh islo ch em ic ala na ly sis w as p erf orm ed u sin g th e a vid in -b io tin -p er ox id as e c om ple x m eth od a s

described previously ( 1 7). The prim ary antibody w as a m onoclonal m ouseantibody raised against human p53 protein and obtained from OncogeneS cie nc e (U nio nd ale , NY). T he a vid in -b io tin -p er ox id as e c om ple x e lite re ag en tand chrom ogen diam inoben/idine w ere from V ector L aboratories, Inc. (B ur-lingam e. C A). Inte nse dark staining w as the crite rion for a positive reaction.S everal term s w ere use d to describe the immunostaining patterns: papillary,w here immunorea ctive cells w ere identified only in the papillary area ; scatte re d, in w hic h o nly s om e iso la te d p os itiv e c ells w er e id en tif ie d; f oc al, w he rec lu ste rs o f p ositiv e c ells w ere se en in so me a re as o f th e e pith eliu m: a nd d if fu se ,in w hich the sheets of positive cells w ere found throughout m ost areas of thele sio ns . I n e ac h e xp erim en t, p ositiv e a nd n eg ativ e c on tro ls w ere in clu de d. T hepositive control was the esophageal carcinom a tissue w hich consistentlyshow ed positive staining in previous expe rim ents. In the negative control,prim ary antibo dy w as not added. T he second negative control w as the esophagea l e pithelium , w hich consistently show ed negative sta ining in previousexperiments.DNA P re pa ra tio n. T is su es f rom s ur gic ally r ese ct ed samp le s w er e c ut in toabout 1-m m1 pieces with a scalpel, washed w ith H,O , and then incubated for4 h at 55 C in a lysis solution (A pplied B iosystem s. Foster C ity, C A) plus

proteinasc K and R Nase A w ith the final conc entration at 250 and l(X ) f ig/m l,re sp ec tiv ely . T he n th e so lu tio n w as s uc ce ss iv ely e xtra cte d w ith e qu al v olu me s3 T he a bb re viatio ns u se d a re: S CC . s qu am ou s ce ll c ar cin om a; B CH . b as al ce ll h yp er

p la si a; S SC P. s in gle s tr an d c on fo rm at io n p ol ym or ph ism; PCR . p olym er as e c ha in r ea cti on .4342

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p S3 AND E SO P HAOEAL C ARC INOGENESI So f T ri s- saturate d p heno l ( pH 8 .0 ):c hlo ro fo rm :i so amy l al co ho l ( 25 :2 4:1 , v /v /v )unt il the p ro te in int erf ac e b ec ame i nv is ib le . DNA was p re cip itat ed w i th e thano land D NA pelle t w as dried and dissolv ed in ste rile w ater. T he concentration ofD NA w as determ ined by spectrophotom etry . W ith the use of b iopsy sam plesand som e resected tum or tissues, areas w ith p5 3 protein acc um ulation w eref irst id entifie d b y immuno staining in o ne o f the slid es. C ells w e re the n sc rap edf ro m the c orre sp ond ing are as in the uns taine d s erial se ctio ns . T he d isse cte dcells w ere dige sted in a ly sis solution containing 50 ITIMT ris (pH 8.0), l mMED TA , 0.5% Tw een 20, and 200 fig/m l proteinase K . The digestion m ixturew as use d d ire ctly in PCR w itho ut furthe r p urific atio n.Poly merase C hain R eaction. O ligonucleo tid e prim ers w ere sy nthesiz edby A merican Sy nthesis. Inc. (Pleasanton, C A). The recom binant Taq DN Apoly merase w as purchased from Perkin Elm er Cetus (Norw alk, CT). De-o xy nuc le oti de s w e re o btai ne d f rom Pharmac ia ( Pis cataway . N J) . T he PTC-HK )

Pro grammab le T he rm al C ontro lle r w as o btaine d f ro m MJ R ese arc h, Inc . Fo urfragments of DNA , including exons 5, 6, 7, and 8 of the p53 gene, w ereamplified. The follow ing primers w ere used: 5'-GTT TCT TTG CTG CCGTGT TC-3'. 5'-A GG CCT GGG GA C CCT GGG CA -3' for exon 5; 5'-TGGTTG CCC AGG GTC CCC AG-3', 5'-AGG AGO GCC ACT GAC AACCA -3' for exon 6; 5'-A CC A TC CTG GCT A AC GOT GA -3', 5'-A GG GGTCAG CGG CAA GCA G-3' for exon 7; and 5'-TTG GO A GTA GAT GGAGCC CT-3'. 5'-A GO CA T A AC TGC A CC CTT GG-3' for exon 8. PCR w ascarried out in 100 /xl of reaction containing 50 mM Tris (pH 9.0), 30 mMMgCK , 100-500 fig of genomic DNA . 40 pmol of each primer, 2(H) JIMc onc entratio ns o f e ac h o f the fo ur d eo xy nuc le otid e trip ho sp hate s. and 5 unitsof Taq poly merase, topped w ith 50 jxl of light m ineral oil. Therm al cy clesc onsisted of an initial 5 m in at 95 Cbefore the addition of T aq poly merase,follow ed by 35 cy cles of 30 s at 94 C ,1 m in at 60 C ,and 30 s at 76 C .PC Rproducts w ere purified from 1.5% agarose gel using the M erm aid kit from B io101 (L a Jolla, C A ).SSC P A naly sis. PC R products of exons 5, 6, 7, and 8 of the p53 gene w erelab ele d at the 5 ' e nd s w ith [7 -'2 P] AT P. N o p urif ic atio n o f the lab ele d p ro duc tsw as re quire d. Fiv e % o f the re ac tio n so lutio n w as run o n ft% p oly ac ry lam id egel (29:1 and 99:1 acry lamide:bisacry lam ide). For exons 6 and 8. threeadditional gel c onditions w ere used: MDE gel (A T B iochem , M alv ern, PA );6% p oly ac ry lam id e ( 99 :1 ); and 1 2% p oly ac ry lam id e (2 9:1 ) w ith 1 0% g ly ce ro l.The gel w as run at 30 W for 8-12 h at room tem perature and cooled w ith a fan.Hum an plac ental D NA (O ncog ene S cience) w as used as norm al control. T hegel w as then dried and exposed to X -ray film (K odak) ov ernight. A ny bandsw ith shifted m obilities com pare d to norm al control w ere c ut out from the geland eluted in H,O at 80 Cfor 10 m in. Fiv e jil of this clute w ere am plified inPCR w ith 20 thermal cy cles using the same pair of primers and the sameconditions as used in the prev ious PC R. The PCR product w as purified andchecked by SSCP analy sis to v erify that the correct bands w ere am plified.W hen a shifted band in SSCP w as detected, the experiment w as alw ay sre pe ate d to c onf irm the re sult.DNA Sequencing. Purified am plified DNA w as directly sequenced bydideoxy chain term ination m ethod. T he se quences of prim ers w ere nested tothe PC R prim ers. Four pairs of prim ers w ere use d to sequence both strands ofDNA . S eq ue nc ing k it w as fro m U nite d S tate s B io chemic al C orp . ( Cle ve land .OH) and p ro ce dure s w e re m od ifie d f ro m the ir stand ard p ro to co l.

12345Fig . 1. R epresentativ e S SC P analy sis of p53 gene exon 5 in esophugeal squam ouscarc ino mas and p rec anc ero us le sio ns . L anes I and 4 , ne gativ e c ontro ls ; L anes 2 and 3 ,no rm al ad jac ent t is sue and t um or o f s am pl e A 9 10 48 7; L ane 5 , b io ps y s am pl e A B 20 2 w it hd ys pl as ia. A rro w s, hand s s ho w ing m ob il ity c hang es .

A910623tumor A910623normalGATC GATC

G>T5'

RESULTSp53 Gene M utation and Protein Accum ulation in Esophageal

Carc in oma. I n o rd er to e stab lish th e r elatio nsh ip b etw ee n p 53 g en emut at io n and p 53 p ro te in ac cumul at io n, e so phag eal c arc inomas w e reanaly zed for mutations in exons 5, 6, 7, and 8 of the p53 gene.R epre sentativ e ex am ple s of S SCP and DNA se que ncing analy se s areshow n in Figs. 1 and 2.Eighteen of the 29 tum or sam ples (62% ) show ed accum ulation ofp53 protein in the nuclei by immunohistochem ical staining. M ost ofthe sam ples had a diffuse staining pattern (T able 1). A m ong these 18staining p ositiv e sam ples, 1 2 had po int m utatio ns includ ing 7 in ex on5, 4 in exon 7, and 1 in exon 8. In one sam ple, in addition to a pointm utation in exon 5, there w as also a 6-base pair insertion in the sam eexon (T able 1). A m ong these point m utations, 9 w ere transitions (7 G

Fi g. 2 . DNA s eq ue nc e anal ys is o f p 5 3 g ene e xo n 5 i n e s op hag eal s quam ous c arc inomaA 91 06 23 and its ad jace nt no rm al tis sue . A rro w, nuc leo tid e s ub stitutio n in co do n 1 58(C GC to C TC ) in tum or.

to A and 2 A to G) and 3 w ere transv ersions (2 G to T and l G to C).D NA sequence analy sis of the 11 /?5J-staining negativ e sam plesre ve al ed 4 s amp le s w i th/;5 .? g ene mut at io ns . The re w e re 2 f ram e- shif tm utations by either a 5-base pair insertion at exon 7 or an 8-base pairdeletion at exon 5. T hese tw o fram e-shift m utations could producet runcat ed p53 p ro te ins due t o p remature t erm inat ion o f the t ransl at io n.T here w ere also 1 silent m utation at codon 136 of exon 5 and 1 pointm utation at intron 5. p53 m utations w ere not detected in the appare ntly no rm al e pithelia ad jace nt to the SCC in the re sec te d eso phag i in

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p S3 AND E SO P HAGEAL C ARC INOOENESI STab le 1 p 53 g ene mut at io n and p ro te in ac cumul at io n i n e so ph ag eal c arc inomasS am ples w ere fro m m ale and f em ale p atients , 4 2- 61 y ears o ld .

Subject9106239104979106749165391682910468910646924114924115911524910514910472917019107439115169104879104735

subjects7subjectsPatlcrn

o f p 53immunostainingDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseFocalScatteredScatteredaaaFocalDiffuseaExon/codon5/1585/1595/1465/5/1737/2377/2397/2458/2805/1755

55/ecep53

mutationBase/aminoacidhangesCGC

.CTC/ArgLeuGCCACC/Ala-.ThrTGG->TAG/Trp-->stop6-basepairnsertionGTG>CTG/Val-LeuATO-ATA/Met-IleAAC>AGC/Ser-PheGGC-GAC/ArgGinAGA'AAA/ArgGysCGC^CAC/Arg-HisCGC-CAC/ArgHisTGC.TTC/CysPheAAC-GAC/AsnAspCAA-.CAG115-basepair insertion,rame-shift8-basepai r de le ti on, f rame-shi f t

" Immunos tain negat iv e .h No a min o a cid ch an ge.' M utatio n no t d ete cte d in ex ons 5, 6, 7 , and 8.

all the 2 9 cases in T ab le I,4 s ug ge sting the m utations o bse rv ed in SCCw e re somatic e vents.In o rde r to e xc lud e the p ossib ility that lac k o f d etec te d m utatio ns atex on 6 o fp 53 ge ne w as cause d by tec hnical b ias in o ur stud y, d iffe rentg el c om po sitions and running c ond itio ns w e re use d in S SCP analy sisof exon 6 w ith a PC R fragm ent of exon 6 containing a point m utationas positiv e control. There w as no mobility shift detected for thesam ple s und er thes e c ond itio ns. D irec t se quencing of PCR pro ducts o fexon 6 dem onstrated the norm al sequence in all these sam ples.pS3 Gene Muta tion a nd P rotein Accumula tion in Esophagea lP recancerous Lesion s. A total of 220 biopsy sp ecim ens of esophageal epithelia w ere obtained from sy mptom -free subjects. M ost ofthem (9 0% ) w e re immuno staining p ositiv e fo r p 53 p ro tein. D iffere ntstaining patterns w ere observ ed and w ere classified as papillary ,scattered, focal, and diffuse. T he results w ere in accordance to ourp re vio us stud y (1 7). T he p erc entag e o f p os itiv e staining ce lls rang edfrom less than 5% (as in A B214) to 75% (as in A B202) of the totalce ll p op ulatio n. G enerally , a hig her p erc entag e o f p5 3-staining p ositiv e cells and a diffuse pattern w ere more frequently observ ed insam ples w ith dy splasia than those w ith low er grade lesions. T hirty -s ev en mmuno st aining pos it iv e s amp le s, i nc lud ing 3 normal e pi thel ia,23 BCH, and 11 dysplasia, w ere analy zed for p53 gene mutation(Table 2). Point mutations w ere detected in 4 of the 11 dy splasiasam ples, 3 of the 23 B CH sam ples, and 1 of the 3 norm al sam ples. A llof these point mutations w ere in exon 5, w ith 3 at codon 175; 2 atcodon 176; and 1 each at codons 159, 135, and 132. There w ere 4transitions (3 G to A and 1 A to G) and 4 transv ersions (3 G to T andl G to C). It w as noted that w hen the percentage of the positiv elystained cells decreased, few er m utations w ere detected am ong thesam ples. For exam ple, m utations w ere detected in 4 of 10 sam plesw hich contained 30-50% p53-staining positiv e cells but in only 2 of22 sam ples w hich contained less than 30% positiv ely stained cells.DISCUSSIONFor the study of the prim ary or early m olecular ev ents of carcino-

g ene si s, b io ps y s amp le s w i th p re canc ero us l es io ns hav e t he ad v ant ag e

ov er cancer tissues; in the latter, m olecular ev ents m ay occur as aconsequence of the neoplasm . T he present study dem onstrates thatp 53 p ro tein ac cum ulatio n o cc urs v ery freq ue ntly in d ysp lasia, a w e llrecogniz ed lesion of esophageal cancer (2, 12, 16). A ccum ulation ofp 53 p ro tein also o ccurs fre que ntly in b asal ce ll hy perp lasia, w hich isco nsid ered as a pre cancero us lesio n b y some inv es tig ators b ut no t b yo the rs b ec aus e o f i ts re ve rs ib il it y. It i s g ene rall y ag re ed , how ev er, thatbasal ce ll p rolife ratio n p re disp ose s the tissue to carcino ge ne sis (1 3-16). p53 protein accum ulation appears to occur am ong these proliferating c ells (17 ). In p relim inary studies , w e fo und that the num be r ofp53 im munostain-positiv e cells increased m arkedly from B CH tody splas ia and incre as ed g reatly fro m d ys plasia to SCC .5 It is p ossib lethat p 53 p ro tein acc um ulatio n is an early ev ent in c arcino ge nesis andthat the p rolife rating ce lls p ositiv ely staine d w ith the p5 3 antib ody inthe papillae are the initiating sites of esophageal carcinogenesis,e sp ec ially if p 53 g ene m utation o ccurs in these ce lls. T he ob serv atio nthat p53 gene m utation w as observ ed in norm al epithelium suggeststhat such m utatio n m ay o ccur ev en be fo re m orp ho lo gic al c hang es c anb e obs erv e d.Am ong the p53-staining positiv e biopsy sam ples, 4 of 11 sam plesw ith dy splasia had point m utation in exon 5 of the p53 gene, w hereasonly 3 had m utations am ong 23 sam ples w ith B CH. It is possible thatour present m ethod w as not sensitiv e enough to detect certain m utations w hen the p53-staining positiv e cells w ere in low abundance. A nattem pt is being m ade to im prov e our scraping m ethod to enrich thep ositiv ely s taine d ce lls fo r m utatio n analy sis. A no the r p ossibility isthat p53 mutation is not the molecular basis for the p53 proteinac cum ulatio n o r is an ev ent sub se que nt to the pro tein ac cum ulatio n insom e of the cases. T hese possibilities rem ain to be exam ined.T he res ults in the current stud y sho w ed a g oo d c orre latio n b etw ee nthe nuclear p53 protein im munostain and p53 gene m utations in 12SC C sam ples, suggesting that the accum ulated p53 proteins in thecancer cells are mostly the mutated forms. There w ere 6 cases,how ev er, in w hich p53 protein lev els w ere high but p53 m utationsw e re no t de tec te d. In thes e 6 cases , it is p ossib le that the acc um ulatio nof p53 protein w as due to a p53 m utation-independent m echanism orthat the p53 m utations did occur but w ere not in exons 5, 6, 7, and 8.T he observ ed m utations are m ost likely som atic ev ents because m utations w ere not observ ed in the 29 sam ples of noncancerous epithelium adjacent to the SC C in the resected esophagi. G erm line m utati ons and p olymorp hisms w i th s ile nt mutati ons hav e no t b ee n re po rte dfo r this p op ulatio n and remain to b e inv estigated .Sev eral comm on features hav e been observed betw een the p53m utation spectra detected in this and other studies (18, 27-30). T hep re v io us ly re po rte d f re que nc y o f p 53 mut at io ns i n human e so phag ealcancer is about 50% and G to A transitions are the predom inant pointm utations (40% of total m utations). S im ilar frequencies of p53 m utations (52% ) and G to A transitions (42% ) w ere seen herein. The Gto A and A to G transitions m ay be a result of carcinogen-inducedalky lation of D NA . T he form ation of O h-m ethy lguanine m ay cause am ispair for thy mine in DNA replication. In the subsequent round ofDNA re plicatio n, an ad enine w o uld rep lace the o rig inal guanine andresult in a G to A transition (31). O n the other hand, the form ation ofO 4-m ethy lthym ine w o uld re sult in an A to G transitio n (1 0, 3 2). T hes em echanism s appear to be consistent w ith the hy pothesis that nitro-sam ines are the c arc ino gens for hum an e so phag eal c arc ino gene sis inH enan, Chi na ( 2, 5 ). A l te rnat iv ely , d eam inati on o f 5 -m ethy lc yt os ineby nitric oxide, w hich m ay be form ed in esophagitis, w ould giv e riseto thymine (33). If this occurs at the noncoding DNA strand, thecoding DNA strand w ould hav e a G to A transition. T he deam ination

4 In s ub je ct 9 10 49 7, t he ad jac ent e pi the lium had d ys pl as ia and a 3 -b as e p ai r d el et io nat ex on 6 of the p53 gene but no po int m utation. ' Unpub li shed resu lt s .

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p S3 AND E SO P HAGEAL C ARC INOGENESI STable 2 p53 gene muta tion and pro tein accumula tion in b iopsy samples from symptom-freesubject s

SubjectAB202AB218C-lC-23AB226AB203AB215AB214C-6AB2283

subjects3subjects2subjects2subjects2subjects6subjects7subjects2subjectsAge

(yr)6338686534502753494021-5948-5047,6632,4443,4622-5731^571,60HistopathologyDysDysDysDysBCHBCHBCHNormalDysBCHDysDysBCHBCo fp53immune-stainingDiffuseDiffuseDiffuseDiffuseDiffusePapillary,

scatteredPapillaryPapillaryPapillaryFocalScatteredDiffusePapillary,

diffusePapillary,scatteredPapillary,focalDiffusePapillaryPapillaryp53

mutationExon/codon5/1765/1755/1595/1325/1765/1755/1355/1acidhangeTOC^TTT/Cys->PheCGC->CAC/Arg->H

*CTC/Arg->LeuTOC^TAC/Cys^TyrCGC-*CAC/Arg-^Hia---

' ,mut at ion not d et ec ted i nexons 5 , 6 , 7 , and 8 .

of 5-methy lcy tosine occurs predom inantly at the CpG site; in thepresent study tw o G to A m utations occurred at C pG sites.In other studies on p53 m utation in hum an esophageal cancers,exons 5, 6, 7, and 8 w ere affected by m utations w ith sim ilar frequencies (18, 27-30). A major difference in our study is that most ofm utations detected herein w ere in exons 5 and 7, w ith only 1 in exon8 and none in exon 6. T his difference in m utation profile m ay reflectthe inv olv em ent of different causativ e agents in the esophageal car-c ino ge ne sis in H enan, Chi na.Int ere sti ng ly , all o f the 8 p5 3 mut ati ons i de nt if ie d i n b io ps y s ampl esfrom symptom-free subjects w ere located in exon 5 and 5 w ere atcodons 175 and 176. T his result suggests a nonrandom p53 m utationpattern during the early stage of esophageal tum origenesis. Thispattern w as not observ ed in the esophageal SCC, in w hich p53m utations w e re m ore d iv ers ely d is tributed and only 2 o f 1 2 m utationsw ere at codon 175. O ne possibility is that codons 175 and 176 of thep53 gene a re within the most susceptible DNA sequences to themutagenesis by particular carcinogens. T he m utations at these tw ocodons m ay render the cells som e grow th adv antage but are not theo nly req uire me nts fo r ne oplastic trans form atio n. T he m utatio ns detected in esophageal SCC m ay have a stronger potential to causeneo plastic trans form atio n b y its elf o r in co op eratio n w ith o ther o nco -g enic p ro ducts. T he pre sent results sug ge st that p 53 g ene m utatio n isan early ev ent in e so phag eal c arcino ge nesis. T his co nce pt ne ed s to b ef urt he r s ub stanti at ed i n s ub se que nt f ol lo w -up s tud ie s.ACKNOWLEDGMENTSWe are grate ful to D rs. C urtis C . Harris and W illiam P. B ennet and to J. A .We ls h o f t he N ati onal Canc er Ins tit ute , f or te chnic al ad v ic e in th e ini ti al p has eo f this w o rk .

REFERENCES1 . Park in, D . M. , Laara ,E. , and Muir ,C. S . Est ima teso f the wor ldwide frequency of 16maj or cance rs i n 1980s . In t. J. Cance r, 4 1: 1 84 -197 , 1 988 .2 . Y a ng , C . S . R es earc h o n e so phag eal c anc er in Chin a: a re v iew . Canc er R es ., 4 0:2633-2644 , 1980 .3 . L ev i, F., O lly o, J., V e cc hia, C . L ., B oy le , P., M onnie r, P., and S av ary , M . T heconsumpt ion of tobacco , a lcoho l and the ri sk o f adenocarc inoma in Barre tt 's esophagus . In t. J. Cance r, 4 5: 8 52 -854 , 1 990 .4 . van Rensburg , S . J. Epi demio lo gi e and d ie tary e v id ence for a s pec if ic nut ri ti onalpred ispos it ion to esophageal cancer. J. Na ti . Cancer Ins t. , 67 : 243-251 , 1981 .5 . Yang , C . S . N it ro samine s and o ther e ti ol og ical f ac to rs i n t he e sophag ea l cance r i nnorthernChina.In: F. N. Magee(ed.), Nitrosaminesand Human Cancer, pp. 487-501.Co ld Spring Harbor, NY: Cold Spring Harbor Labora tory , 1982 .6 . S inger, G . M. . Chuan, J. , Roman.J. , and Linj insky ,W . Ni trosaminesand nit rosamine

10 .

12 .13 .14 ,

15 .16 .17 .

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p53 tumor suppressor gene mutation in early esophageal

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