Cancer Res-1991-Tamura-3056-8_2

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1991;51:3056-3058. Published online June 1, 1991.Cancer ResGen Tamura, Toshimasa Kihana, Kazuhiro Nomura, et al.Single-Strand Conformation Polymorphism AnalysisCancer by Cell Sorting and Polymerase Chain Reaction

Gene Mutations in Primary Gastricp53Detection of Frequent

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( CA NCER RES EA RCH 5 1. 3 05 6-3 05 8. J un e I. 1 99 1]

A dvances in B riefDetection of Frequent p53 Gene Mutations in Prim ary Gastric Cancer by CellS ortin g and Polymerase Chain Reactio n S ingle-S trand Con fo rmationPolymorphism Analysis 'G en T annini. T oshim asa K ihana, K azuhiro N omura, M asaaki T erada, T akashi Sugim ura, and Setsuo H irohashi2D iv is io ns o f P at ho lo gy G .T ., T . K ., S . H .l , N eu ro su rg er y K .N .] a nd G en et ic s [ M. T ., T . S .] , N ati on al C an ce r C en te r R es ea rc h In sti tu te a nd H os pi ta l, 1 -1 ,T su ki ji 5 -c ho me , C hu o-k u, T ok yo 1 04 , J ap an

AbstractMuta tio ns o f th e p 53 g ene w ere in ve stig ate d after tu mor ce ll en ric hmen t b y c ell s or ti ng b as ed o n d if fe re nc es in DNA con te nt a nd p ol ymer as ec ha in re ac ti on s in gl e- st ra nd c on fo rm at io n p ol ymor ph ism a na ly si s i n 2 4surgical specim ens of prim ary gastric cancer. p53 m utations w ere detected in exons 4-8 in 64% (9 of 14) of aneuploid tum ors but in none of1 0 d ip lo id t umor s e xami ne d. F ou r o f f iv e t umo rs c on ta in in g two o r t hr eean eu plo id su bp opu lation s sh ow ed th e p re sen ce o f p 53 g en e m utatio ns.N o c or re la tio n was f ou nd b etw ee n t he p re se nc e o f p 5 3 mut at io ns a nd th ed eg re e o f h is to l gi ca ! di ff er en ti at io n o f t umo rs . Th es e f in di ng s s ugge stthat p53 gene m utations are related to D NA ploidy alterations as relat iv el y l at e e ve nts o f c ar cin og en es is in g as tri c c an ce r. T he p re se nt m eth odi s h ig hl y s en si ti ve f or d et ec ti on o f g en et ic a bnormal it ie s a nd i s a pp li ca bl eeven w hen various kinds of nontum orous cells are present in tum orsamples.

IntroductionT he recen t pro gress m ad e in m olecu lar ge netics h as re vealeda co nsisten t set of g ene tic alteratio ns in v ario us human can cerspossibly corresponding to m ultistep tum or developm ent. G astric cancer is no exception, and m ultiple genetic alterationsincluding ras oncogene m utations, gene am plifications, andchromosomal loss of heterozygosity have been detected, al

though the incidence of these alterations is low compared tothose in colorectal carcinoma, another major cancer of thegastrointestinal tract (see Ref. l for review ). R ecently, it hasbeen shown that the p53 gene is a tumor suppressor gene (2)and that its mutations play an important role in the development of many common human malignancies (3). In gastriccancer, how ever, p53 gene m utations have not been detected inth e p rim ary site, hav in g b een d em on strate d on ly in m tastase sand cell lines by use of ordinary D NA extraction proceduresan d the p olymerase ch ain reactio n (1 ). A ltho ug h these fin din gsseem to suggest that mutations of the p53 gene are a very lateevent in gastric carcinogenesis, it is also possible that suchm utations could have been underestim ated in surgical specim en s of p rim ary g astric can cer, w hich are often h eav ily co ntaminated w ith norm al strom al cells and inflam matory cells. A ccordingly, we used cell sorting based on differences of DN Acontent, follow ed by PC R'-SSC P analysis (4), to detect basechanges in p53 gene sequences (5).

Rece iv ed 3 /4 /9 1; a ccept ed 4 /5 /9 1.T he costs of pub lic ation o f this artic le w ere de fraye d in part by the paym ento 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 ar ke d a dv ertis em en t ina cc or da nc e w ith 1 8 U .S .C . S ec tio n 1 73 4 so le ly to in dic ate th is fa ct.1 Th is w ork w as su pp orte d in p ar t b y a G ra nt-in -A id fo r a C om pr eh en siv e 1 0-Year Strategy for Cancer C ontrol from the M inistry of Health and W elfare,Japan.2 T o w hom re qu es ts fo r r ep rin ts sh ou ld b e a dd re ss ed .3 T he a bb re via tio ns u se d a re : P CR , p oly me ra se c ha in r ea ctio n; S SC P. s in gle -s tr an d con fo rma ti on pol ymo rphi sm .

M aterials and M ethodsDNA flow cytometry and cell sorting using a FACS-IV (BectonD ic ki ns on , Mou nt ai n V iew, CA ) was p er fo rm ed o n 2 4 f ro ze n s pe cime nsof prim ary gastric cancer surgically resected at the N ational C ancerC en te r H osp ita l, T ok yo , Jap an. N uc lei w ere iso lated w ith 0 .2% T rito nX -1 00 (S igma C hemica l C o., S t. L ou is , MO ), trea te d w ith 0 .1% RNa se(S igma), sta in ed w ith 5 0 ug /m \ p ro pid iu m io did e (S igma), a nd filte re dthrough nylon m esh. A neuploid tum or cell populations w ere sortedwhen determ ined, and the cells at S + G2M in diploid tumors weresorted for tum or cell enrichm ent. T hen all the exons of the p53 genew ere am plifie d d ire ctly fro m 1 0' so rte d n uclei b y th e p olyme rase ch ain

r ea cti on ( PCR ) u sin g s pe ci fic o lig on uc le oti de p rime rs a s s hown in T ab le1. T he P CR products w ere subjected to single-strand conform ationp olym orph ism (S SCP) a naly sis, a ne wly de velo pe d m eth od for d ete ction of structural alterations in D NA including point m utations (4). Asecond P CR -S SC P analysis w as perform ed to ensure that the resultsw ere reproducible in each case w hich show ed m obility shift. For sequencing analysis, exon 5 or 7 of the p53 gene from 4 tumors withm obility shift detected by S SC P analysis w as am plified by P CR . T hePC R products w ere cloned into pU CIS vector. A bout 50-100 m ixedcolonies were am plified and then sequenced by the dideoxy chainte rm in atio n m etho d (6 ). T he p rim ers u sed fo r se que ncin g w ere s en se ,5'-T CT TCCTGCAG TA CTCCCT -3' and antisense, 5'-A GC TG CT -CACCATCGCTA T-3' for exon 5, and sense, 5'-ACTGTACCAC-CATGCACTAC-3' an d a ntise ns e, 5 '-G TGGCTCCTGACCTGGA -G TC-3' for exon 7.Results

DNA flow cytometry proved that 14 (58%) of the tumorsw ere aneuploid and the other 10 (42%) were diploid. Two orth ree an eu ploid su bpo pu latio ns (m ultip lo id y) w ere d etected ina sample in 5 of the 14 aneuploid tumors (Fig. 1). M utationsof the p53 gene were detected in exon 4 in one tumor, exons5-6 in three and exons 7-8 in five by PC R-SSC P analysis (Fig.2). A s w ell as tw o bands w ith m obility shifts, tw o bands corresponding to the norm al al elew ere seen in four tum ors but notin the rem aining five. It could not be determ ined w hether tw obands corresponding to the norm al al elew ere from norm alcells still present after cell sorting or from the rem aining w ild-ty pe al eleo f tumor cells in th e fou r tumo rs. A ll th e m utatio nsof the 53 gene were detected in aneuploid tumors, but none indiploid tum ors (Table 2). Four of five m ultiploid tum ors contained p53 gene m utations, and different aneuploid subpopulations from a tum or sam ple show ed the sam e m obility shift byPC R-SSC P analysis (Fig. 2) in all four cases. N o correlationw as found betw een the presence of p53 gene m utations and thedegree of histological tumor differentiation (Table 3). Themutations of the 53 gene, confirm ed by sequencing analysis,w ere point m utations at the third position of codon 173 (G TG -GTA) and at the second position of codon 251 (ATC-AGC)(F ig. 3), a 7-base pair deletion at the first position of codon 137

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p 53 GENE MUTATIONS IN GASTRI C CANCERT ab le 1 O lig on uc le oti de p rim e rs (5 '3' )Exon

Upstreamownstream1GGA A TTCGGA TTCCTCCA A A A TGA TTT GGA A TTCCA GTCA GGA GCTTA CCCA2 GGA A TTCTGGA TCCTCTTGCA GCA GCCA GGA A TTCCA A TGGA TCCA CTCA CA G3 GCTCTTGA CTTTCA GA CTT GGA A TTCA A CCCTTGTCCTTA CCA GA A4 GGA A TTCTTTTCA CCCA TCTA CA GTCC GGA A TTCCTCA GGGCA A CTGA CCGTGC5-6 GGA A TTCCTCTTCCTGCA GTA CTCC GGA A TTCA GTTGCA A A CCA GA CCTCA7-8 GGA A TTCTCCTA GGTTGGCTCTGA C GGA A TTCCTGCTTGCTTA CCTCGCT9 GGA A TTCTTGCCTCTTTCCTA GCA GGA A TTCCCCA A GA CTTA GTA CCTG10 GGA A TTCCTCTGTTGCTGCA GA TC GGA A TTCGCTGA GGTCA CTCA CCT1 GGA A TTCTGTCTCCTA CA GCCA CGAATTCTGACGCACACCTATTGCmax

.IKJ13

I;, iif i 'i i'v '....J0

" " '. " - - y v V . .' "' \ T ab l e

3 p 53 g en e m u tati on an d h is to lo gic al t yp e i n p rim ary g as tricancerp53geneutationHistological

ty pe" Positiv e N egativ eotalW elldif ferentiated 5 7 12 I tUndif ferentiated 4 829

154"SeeR ef . l.* N S , n ot s ig ni fic an t b y Fis he r'ses t .ACGTm0

Channel N o. 250F luorescence In tens ityFig . 1 . D N A h is to gram o f a m ultip lo id tu m or. T w o an eu plo id p eak s (arro ws)appear to the right of the norm al diploid peak . A neuploid populations w eres ep arate ly s orte d an d s ub je ct ed t o PC R -S S CP an aly sis .

]codon 25112345 Fig . 3 . S eq ue nc in g au to rad io gram o f e xo n 7 o f th e p 53 g en e. A p oi nt m u tat io n,T to G transition , is present at the second position of codon 251 (A T C to A G C).

T ab le 4 M u tatio ns o fp 53 g en e in p rim ary g astric can cerExon5

577Codon137-13973251252-253Nucleotide

substitution7-basep ai r d el et io n ( CTGGCCA )GTG to GTAA TC to A GC4 -b as e p ai r i ns ert io n ( CTCA )

-to the f irst position of codon 139 (CT GG CCA ), and a 4-basepair insertion at the f irst position of codon 252 to the f irstposition of codon 253 (C TC A ) (T able 4).

Fig. 2. S SC P analy sis of ex ons 5-6 of the p53 gene in gastric cancers. L ane I,n orm al g as tric m uco sa; L an es 2 -5 , g astric can cers w ith m ob ility sh if ts. L an es 2an d 3 are f ro m d if f ere nt an eu plo id s ub po pu lati on s o f a t um o r s am p le .

T ab le 2 p 53 g en e m utatio n an d D N A p lo id y in p rim ary g as tric can cerDN AloidyDiploidAneuploid(Multiploid)p53

genePositive0949mutationNegative105115Total10

L14 5!24

1P


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p 5 GE NE MUT AT IO NS I N G AS TR IC C AN CE Rprogression (8); the rem aining al eleof the p53 gene w as considered to hav e been deleted in at least 56% (5 of 9) of thetum ors. In contrast, no diploid tum ors contained p53 genem utations. T his is v ery dif f erent from the situation in raso nc og en e ac tiv atio n; mu tatio n o f th e ras o nc og en e is c on sid ere dto be an early ev ent in carcinogenesis and is present in diploidcells f ro m w h ich an an eu plo id sub po pulatio n arises (7).V ery recently , it has been suggested that m utations of p53are the rate-lim iting step in its inactiv ation and that once amu tatio n o cc urs , lo ss o f th e remain in g w i ld -ty pe al elerap id lyf ollow s, based on the f inding that the great m ajority of colonietum ors containing p53 gene m utations show loss of this w ild-ty pe al ele(9). In addition, deletion of chrom osom e 17p m ayoccur sim ultaneously w ith m any other chrom osom al lossesthro ug h ab no rm al m ito sis (1 0). T hese an d o ur p resen t f in din gssu ggest thatp5 3 g en e m u tatio ns in common m alig nan cies o ccu ras re lativ ely late e ve nts in c arc in og en esis an d th at c hromosomalinstability , w hich m ak es cancer cells aneuploid, play s an important role in the selection of tum or cells w ith p53 genem utations by causing easy loss of the rem aining al eleandto tally in ac tiv atin g th e n ormal p 53 f un ctio n.In m ultiploid tum ors, dif ferent aneuploid subpopulationsf rom a tum or sam ple show ed the sam e m obility shif t by PCR -S SC P analy sis, indicating the presence of identical p53 genem utations. T herefore, p53 gene m utations precede the ev olution of dif ferent aneuploid subpopulations, w hich are deriv edf rom a c ommon an eu plo id an ce stral c lo ne , c au se d b y ad ditio nalc hromosomal e ve nts.A lthough no correlation w as found betw een the presence ofp53 gene mutati ons and the hi stol ogi cal gr ade of tumor di fferen tiation in ou r stu dy , f urther clin ico path olo gical stu dies w ill

b e n ece ssary in o rd er to elu cid ate th e p ro gn ostic sig nif ican ce o fp53 gene mutations.Acknowledgments

W e thank K . Iw asak i. D iv ision of N eurosurgery . N ational C ancerC en te r, T ok y o, Jap an , f or h er tec hnical h elp in ce ll so rtin g.References1 . H iro has hi. S ., an d S u gim u ra. T . G en et ic al te rati on s i n h um an g as tri c c an ce r.C an ce r C el ls . 3 : 4 9 -5 2. 1 99 1.2. Finlay , C . A ., H inds, P. W ., and L ev ine, A . J. T he p53 prolooncogene canac t as a s up pre ss or o f t ran sf orm at io n. C el l. 5 7: 1 08 3-1 09 3. 1 99 0.3. N igro. J. M ., B ak er. S . J.. Preisinger. A . C.. Jessup. J. M .. Hosteller, R .,C leary , K ., B ig ner. S . H .. D av id so n. N ., B ay lin . S .. D ev ilee. P.. G lo ver, T .,Collins. F. S ., W eston, A ., M odali. R ., H arris. C . C ., and V ogelstein, B .M utations in the p53 gene o ccur in div erse hum an tum our ty pes. N ature(L o nd .) . 3 42 : 7 05 -7 08 . 1 98 9.4. Orila. M ., S uzuk i. Y .. S ek iy a. T .. and Hayashi, K . R apid and sensitiv ed et ec ti on o f p oin t m u tat io ns an d DNA p ol ym o rp his m s u sin g th e p oly m eras ec hain re ac ti on . G en om i cs , 5 : 8 74 -8 79 . 1 98 9.5. B uchm an. V . L .. Chum ak ov , P. M .. N ink ina. N . N .. S am arina. O . P., andG eo rg iev . G . P. A v ariatio n in th e stru ctu re o f th e p ro tein -co din g reg io n o fth e h um an p 53 g en e. G en e, 7 0: 2 45 -2 52 . 1 98 8.6. S anger, F.. N ick len. S .. and Coulson. A . R . D NA sequencing w ith chain-te rm i nati ng i nh ib ito rs . Pro c. N at i. A c ad . S ci . U S A , 7 4: 5 46 3-5 46 7, 1 97 7.7. B urm er. G. C ., and L oeb. L . A . M utations in the K RA S 2 oncogene duringp ro gressiv e stag es o f h um an co lo n carc in om a. Pro c. N ati. A c ad . S ci. U S A ,8 6: 2 40 3- 24 07 . 1 98 9.8. Hattori. T .. S ugihara. H.. Fuk uda. M .. H am ada. S ., and Fujita. S . DN Ap lo id y p attern s o f m in ute carcin om as in th e sto m ach . Jp n. J. C an cer R es .,7 7:2 76 -2 81 . 1 98 6.9 . B ak er. S . J.. Preis in ger. A . C .. Jessu p. J. M ., Parask ev a. C .. M ark ow itz , S ..W i lls on . J. K . V . . H am ilto n. S .. an d V o gelstein . B . p 53 g en e m utatio ns o ccu rin c om b in at io n w ith 1 7p all eli c d el eti on s as l at e e ve nt s i n c ol ore ct al tu m ori -g en es is . C an ce r R e s. . 5 0: 7 71 7-7 72 2, 1 99 0.10. V ogelstein, B .. Fearon. E. R .. K ern. S . E.. H am ilton. S . R .. Preisinger, A .C ., N a kamu ra. Y . , a n d W h it e. R . A l le lo ty p e o f c ol ore ct al c arc in oma s. S c ie nc eW a sh in gt on DC ). 2 44 : 2 07 -2 11 . 1 98 9."

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Cancer Res-1991-Tamura-3056-8_2