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1998;4:1071-1074. Published online April 1, 1998.Clin Cancer ResH Ouyang, T Furukawa, T Abe, et al.endometrium.genetically unstable cancers of the colorectum, stomach, andThe BAX gene, the promoter of apoptosis, is mutated in

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1072 Som atic M utations ofBAX in M I+ C ancers

Ta b le 1 Sum m ary of m utations detected inBAXHi s t o l o g i c a l

d i a g n o s i sand clinical N o. of N o. of N o. ofBAX

Tum ors stagea tum ors M I+ cases m utationsE ndo m etrial cancer

G1G2G3C learSerousStage IStage IIStagelllStagelV

10048292021

6516181

26118610

19340

3111002010

Gastric cancer

C olonectal cancerb

IntestinalDi f f u s eO thersStagelStagellStage II IStagelVW ellM odPoorM ucD ukes AD ukes BD ukes CD ukes D

82353 89

27123211

1184041334

51104512

157445352

2240

1718293

52121121910712151

Pancreatic cancer 60 9 0e n d o me t n i o i da G 1, endom etnioid adenocarcinom a grade 1;G 2,

adenocarcinom a grade 2; G 3, endom etrioid adenocarcinom a grade 3;clear, clear cell adenocarcinom a ; serous, serous adenocarcinom a ; intes-tinal, intestinal type of gastric cancer; diffuse, diffuse type of gastriccancer; w ell, w ell-differentiated adenocarcinom a; m o d, m o derately dif-fenentiated adeno carcino m a; poo r, p oorly differentiated adeno carcino -m a ; m u c, m u cinous adenocarcinom a.

b Tw o of the 1 18 colorectal cancers w ere developed in I H N PC Cpatient.

M utation Ana lyses ofB A X and ICE . M utations ofBAXand IC E at the repetitive sequences w ithin the coding regionsw ere determ ined by a PC R -based assay (10). In brief, each15-pA reaction m ixture [10 ng of D N A , 6.7 m ivi Tris-H C 1 (pH8.8), 16.6 m M (N H 4)2SO 4, 10 m i 3-m ercaptoethano1, 6.7 p.MEDTA , 6 .7 m M M gC l2, 0.33 m ivt of [-y-32P]A TP-labeled andunlabeled prim er, 1.5 m i each deoxynucleotide, 10% (v/v)DMSO , and 0.75 unit of Taq D N A polym erase] w as am plifiedfor 40 cycles w ith the follow ing regim e: denaturation at 94#{176}Cfor 30 5; annealing for 30 s at55#{176}Cfor the poly(C )6 tract ofB A Xand the poly(A )8 tract ofICE and at 58#{176}Cfor the poly(G )8 tractof BAX; and extension at 72#{176}Cfor 30 s. R epetitive sequencesw ithin the ceding region of theBAX gene [the poly(G )8 andpoly(C )6 tracts at nucleotides 114-121 and 260-265, respec-tively] and the ICE gene [the poly(A )8 tract at nucleotides2084-2091] w ere analyzed. N ucleotide sequences of the prim -ers w ere as follow s: 5-U CA TC CA G GA TC GA G CA G G-3and 5-A C TC G C TC A G C TFC U G G TG -3 for the poly(G )8tract in B A X ; 5-A TG A TFG C C G C C G TG G A C A -3 and 5-C A G TFG A A G TFG C C G TC A G A -3 for the po!y(C )6 tract in

A

WI . . --III.-

TNTNTNTNTNDNCC37 C 36 G47 G 51 E68 C 31

B

F ig . I A, screening for m u tations of the poly(G )8 tract (an eight-deoxy-guam n e repeat) in theBAX gene. D eletions of 1 bp in a colorectal cancer(C36) and gastric cancers (G47and G51 ) are show n . B oth the l-bp deletionand the 1-bp insertion w ere show n in descending colon(D ) and cecum (C )cancers of one FIN PC C patient and in endom etrial cancer case E68,w hereas the 1- and 2-bp deletions w ereobserved in a colorectal cancer(C 37). T and N denote D N A S fromtum orsand the corresponding norm altissues, respectively. W, + 1 , -1 , and -2 denote the w ild type, l-bpinsertion, and 1- and 2-bp deletions, respectively.B , nucleotide sequencingan aly sis at the poly(G )8 tract of the BAX gene in E6 8 (endometrial cancer),G4 7 (gastric cancer), andC37(coborec ta l cancer). N u cleotide sequences ofthe sense strands are show n. D eletions of 1 and 2 bp and an insertion ofbp at the poly(G )8 tract are indicated bya r r owh ead s . The nucleotidesequence of the w ild type(W) is also shown.

B A X ; and 5-A TC TG G G G TA C A G C G TA G A T-3 and 5-TA -G A A C TFA C C G A A G C A G TG A -3 for the poly(A )8 tract inICE . M utations w ere confirm ed by nucleotide sequencing anal-ysis according to the m ethods described previously (10).

RESULTSW e first exam ined BAX for m u tations at repetitive sequences

w ithin its coding region in 360 hum an tum ors of4 different organs,as sum m arized in Table 1. Tw o regions of the gene, an eighguanine stretch [the poly(G )8 tract at nucleotides 114-121] andsix-cytosine stretch [the poly(C )6 tract at nucleotides 260-265],w ere analyzed. Typical exam ples are show n in Fig. 1A . C olorectalcancer case C 37 show ed 1- and 2-bp deletions, w hereas C 31D andC3 1C , cancers that had developed in a single FIN PC Cpatient,show ed a 1-bp insertion and a l-bp deletion at the poly(G )8 m irosateffite w ithin BAX. These tum ors had tw o-hit m utations; thew ild-type band w as presum ably contributed by contam inating norm al cells. Sim ilarly, in case E68, anendom etrial cancer, atw o-hitm utation w as observed; a 1-bpinsertion as w ell as a l-bp deletionw as clearly seen. G astric cancer cases 047 andG 5 1 and colorectal

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C linical C ancer R esearch 1073

Ta ble 2 Sum m ary of m utatio ns detected in BAX , IGFIIR , and RI!

Tum ors C ase no.H istologicaldiagnosis

C linicalstage

M utationsBAX IG F IIR RI! M I

Colorectal cancer C 23C31CC A 3 6C IPC 4C SC 7C 8C lOC 22C 29C 3IDC 36C 37C 62C 63

W ellW ellM odPoorPoorPoorPoorPoorPoorPoorPoorPoorPoorPoorPoorM uc

D ukes AD ukes AD ukes AD ukes CD ukes CD ukes DD ukes AD ukes AD ukes CD ukes DD ukes BD ukes CD ukes CD ukes CD ukes AD ukes C

-

+-

--

+-++-++++-

+

-

-

-

NDN D

-

NDN D

-

--

+-

-

-

-

+++++++++++++++-

++++++++++++++-

+Gastric cancer

E ndom etrial can cer

G 28G 34G 48G 84G l05G 139G lG 37G 41G 5 1G l8G 35G 47E87EllSE517ESlOE90E94E68

IntestinalIntestinalIntestinalIntestinalIntestinalIntestinalD iffuseD iffuseD iffuseD iffuseO thersO thersO thersG 1G ,G ,G 1G2G 2G 3

S tage IIIStage IStage IStage IStage IS tage IIIS tag e IIIStage IS tage IVS tag e IllS tage IllS tage IIS tage IVStage IStagelStagebS tage IIStagebStage!Stagell!

--+-

-

+-

--

+-

++-+-

-

-++

-

-

-

+-

+-

-

-

-

-

-

++-

+++-

-

+++++-

+++-

+-

+--

-

-

--

-

++++++-

+-+++++++++++

a G 1, endom etrioid adenocarcinom a grade 1; G2 , endom etnioid adenocarcinom a grade 2; G 3, endom etrioid adenocarcinom a grade 3; clear, clear celladenocarcinom a; serous, serous adenocarcinom a; intestinal,intestin al typ e of g astric cancer, diffuse, diffuse type of gastric cancer; w ell, w ell-differentiatedadenocarcinom a ; m o d, m o derately differentiated adenocarcinom a ; poor, poorly differentiated adenocarcinom a ; m u c, m u cinous adenocarcinom a .

b , m utations w e re detected; - m u tations w ere not detected; N D , not determ ined.C Tw o of the 118 coborectal cancers, C 31C and C 31D , w ere developed in I H N PC C patient.

cancer case C 36 show ed l-bp deletions. These results are sum m a-rized in Tables 1 and 2. N o m utation w as observed in any of thepancreatic cancers.

T o confirm that these alterations occurred in the repetitivepoly(G )8 region of each PC R product show ing m utation, thenucleotide sequences of these tum ors together w ith D N A s fromtheir corresponding norm al tissues w ere analyzed. Exam ples areshow n in Fig. lB . D N A from endom etrial cancer E68 gainedone guanine residue in the poby(G )8 region ofBAX, w hereas ingastric cancer G 47, a l-bp d e le tion in the p o ly (G )8 tra ct w asclearly observed. Sim ilarly, in C 37, both 1- and 2-bp deletionsw ere observed. W e further subcloned the PC R product of tum orD N A C 37 in the EcoR V site of pB luescript II SK (+) anddeterm ined the nucleotide sequences of the 40 independentclones: the num ber of clones containing poby(G )8, poly(G )7, andpoly(G)6 w as 18, 1 1, and 1 1, respectively.

W e found no m utations in the poly(C )6 tract of theBAX geneor in the poly(A )8 tract of theI CE gene (data not show n). W e

further screened M I- cancers in these organs, but again, no m u-tations w ere found (data not show n). In conclusion, m utation of theBAX gene w as observed in three endom etrial, five gastric, and ninecolorectal cancers w ith M I. N o correlation betw eenBAX m utationand age, stage, or histological type w as observed. Tum ors in w hichm utations of the BAX gene w ere observed are listed in Table 2 w ithour previous results forI G F - I I R and RI! (8, 10).

DISCUSS IONR ecently, M I has been found to play a significant role in

various hum an tum ors (1-4). M I+ tum or cells tend to havem utations in repetitive sequences throughout the genom e (26),and frequent m utations w ere reported inRI ! and IGF-I IR inM I+ can cers (8 -10 ). BA X is a BC L -2 -re la ted pro te in th a tprom otes apoptosis. M utation ofBAX w as reported in cell linesderived from hem atological m alignancies (27). R ecently, fre-quent fram eshift m utations ofBAX in the m icrosatellite w ithin



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1074 S om atic M u tation s of BAX in M I+ Cancers

th e cod ing sequ ence w ere reported in som e cell lines and p ri-mary co lon tum o rs (1 1 ). In the presen t s tudy , w e observ edsom atic fram esh ift m u tations in th e po ly (G )8 m icrosa te llite o fBAX in M I+ endom etria l (3 of 26 cases , 12% ), gas tric(5 of 15cases, 33% ), and co lo rec ta l (9 o f 22 cases, 4 1 % ) cancers;fram esh ift m uta tio n shou ld resu lt in the prod uc tio n of trun ca tedB A X p ro te in . T hus , our p resen t resu lts sug gest tha t: (a ) m u ta -tion s of the flAX gene p lay an im por tan t ro le in the genesis o fen dom etria l, gas tric , and coborec ta l cancers w ith M I; an d(b ) th eBAX gen e is on e of the ta rge t gen es fo r gene tic a lte ra tion s ingas tric , c ob ore ctal , an d endom etr ia l cance rs w ith M I.

A lth ough the inc idence o f M I is h igh in p an creatic can cer,w e d id no t ob serve any m utation inBAX in 60 pancrea ticcan cers , 9 o f w hich (15% ) w ere M I+ . W e prev ious ly s tud iedm u ta tions of RI! an d IGF-IIR and fo und no m uta tio ns in pan-crea tic cancers . T here a re possib le exp lana tio ns fo r th is find ing :(a) B A X does not play an im portant rolein pan cre atic carc ino -g en esis ; o r (b) m uta tions in o th er reg ions of the gene are c ru cia lin pancrea tic ca rc inogenes is. B ecause repe titive sequences asana lyzed in th is s tu dy are frequ en t ta rge ts fo r m uta tio n ing en e tica lly uns tab le ce lls , w e suspec t th at the fo rm er p oss ib ilityis m ore like ly than the la tte r. Th ere m ay b e a gene(s ) o the r thanBAX, RI!, or !GF-I !R tha t p lays an im portan t ro le(s) in p ancre -a tic ca rc ino genesis. A dd itio na l s tud ies a re n ecessa ry to id en tifyo ther p oss ib le ta rge t gene(s) o f M I in th is tum or type to u nder-s tand the co urse of ca rc ino genesis.

A C K N O W L ED G M E N TSW e thank H inom i 0 . S h iw aku for assistance in prepara tion of the

manuscr ip t .

R E F E R E N C E S1 . A alto nen , L . A ., Pe ltom ak i, P ., L each , F . S ., S is tonen , P ., R y lkkanen ,L ., Meckl in , J . P ., Ja rv in en , H ., P ow ell, S . M ., Jen , J ., H am ilto n , S . R .,P e te rsen , G . M ., K inz ler , K . W ., V oge lste in , B ., and d e la C hape l!e , A .C lues to the p atho genesis o f fam ilia l co lo rec ta l cancer. S c ience (W ash -i n g t o n DC) , 2 6 0 : 8 1 2 - 8 1 6 , 1 9 9 3 .2. Thibodeau , S . N ., B ren , 0 ., an d Scha id , D . M icrosate llite instab ilityin cancer o f the prox im al co lon . Sc ience (W ash in g ton DC ), 260: 816-8 1 9 , 1 9 9 3 .3 . Ion ov , Y ., Pe inado , M . A ., M abkhosyan , S ., S h iba ta , D ., and P eru cho ,M . U biq uitous som atic m u tat ion s in s im ple rep eated seq uen ces revea l anew m echan ism for co lon ic ca rc ino genesis. N a tu re (L ond .),363: 558 -5 6 1 , 1 9 9 3 .4 . H an , H -J., Y anag isaw a , A ., K a to , Y ., P ark , J-G ., and N akam ura , Y .G ene tic instab i lity in p anc rea tic c anc er and poor ly d iffe ren tia ted type o fga str ic c anc er. C ance r R es. ,53 : 5087-5089 , 1 993 .5 . M ark ow itz , S ., W ang , J ., M yero ff, L ., P arson s, R ., S un , L ., L u tten -baug h , J ., F an , R . S ., Z borow ska , E ., K inz le r, K . W ., V og els tein , B .,B ra tta in , M ., an d W illson , J . K . V . Inac tiva tion of th e typeII T G F -3recep to r in co lo n cancer ce lls w ith m icro sa te llite ins tab ility . S c ien ce(W ash ing ton DC ), 268: 1 3 3 6 - 13 3 8 , 1 9 9 5 .6 . Parsons , R ., M yero ff, L . L ., L iu , B ., W illson , J . K . V ., M ark ow itz ,S. D ., K inz le r K . W ., and V og els tein , B . M icrosa te llite in stab ility an dmu tat ion s o f th e tra nsform ing grow th fa cto r 3 typ e II re cep tor g en e inco lo rec ta l cancer. C ancer R es .,55 : 5548-5550 , 1995 .7. Meyero ff, L . L ., Parsons . R ., K im , S-L ., H ed iick , L ., C ho , K . R ., O rth ,K ., Math is , M ., K inz le r, K . W ., L uu erbaugh , J., P a rk , K ., B ang , Y -J ., L ee,H . Y ., Park , J-G ., Lyn ch , H . T ., R oberts, A . B ., V ogebs te in , B ., andMarkow itz , S. D . A transfo rm ing g row th fac to r 3 recep to r typeII genem uta tio n comm on in co lon and gastric b u t ra re in end om etrial cancers w ithm icro satelli te instab il ity . C ance r R es. ,55 : 5545-5547 , 1995 .

8 . A be , T ., O uyang , H ., M ig ita, T ., K a to , Y ., K im ura, M ., Sh iiba , KSunam ura , M ., M atsun o , S ., and H on . A . T he som atic m uta tion frqu en cy of the transfo rm ing grow th facto r 3 recep to r typ e II gene variesw ide ly am ong d iffe ren t c anc ers w ith m icro satelli te in sta b il ity . EJ. S urg . O ncob ., 22 : 474-477 , 1996 .9. Souza , R . F ., A ppe l, R ., Y in , J ., W ang , S ., Sm olin sk i, K . N ., A b ra -ham , J . M ., Z ou , T -T ., Sh i, Y -Q ., L e i,J., Cottre l, J., C ym es , K ., B iden ,K ., Simm s, L ., L egg ett, B ., L yn ch , P . M ., F raz ie r, M ., Pow ell, S . MH arpaz , N ., Su g im ura , H ., Y oung , J ., an d M elize r, S . J. M icrosa te lliteins tab ility in the insu lin -like grow th fac to r II recep to r gene in gas tro in -testina l tum ou rs. N a t. G ene t., 14 : 255 -25 7 , 19 96 .1 0 . O uy ang , H ., Sh iw aku , H . 0 ., H agiw ara , H ., M iu ra , K ., A b e,K ato , Y ., O h tan i, H ., S h iib a, K ., Sou za , R . F ., M eltze r, S . J., and H on .A . The in su lin -like grow th facto r II recep to r g en e is m uta ted in gen et-ically uns tab le cancers o f th e endom etn ium , stom ach , an d co lo rec tum .C an cer R es., 57 : 1851-1854 , 19 97 .11 . R am pino , N ., Y am am o to , H ., Ionov , Y ., L i, Y ., S aw ai., H ., R eedJ. C ., an d Peru ch o , M . S om atic fram esh ift m uta tio n in th eBAX gene inco lon can ce rs of the m icro sate llite m uta to r ph eno typ e. Sc ien ce (W ash-ing ton D C ), 275 : 967 -969 , 1997 .12 . O ltva i, Z . N ., M illim an , C . L ., and K o rsm eyer, S . J. B c l-2 herod imer izes in vivo w ith a con served hom o log , bax , tha t a cce lera tesprog ramm ed ce ll dea th . C e ll, 74 : 6 09-61 9 , 1993 .13 . M iy ash ita , T . , K rajew sk i, S ., K rajew sk a, M ., W ang , H . G ., LH . K ., L ieb erm ann , D . A ., H offm an , B ., and R eed , J . C . T um or suppressor p5 3 is a regu lato r o fbcl-2 an d bo x g en e e xp re ss io n in vitro an din vivo . Oncogene , 9: 1799-18 05 , 19 94 .14 . M iy ash ita , T ., and R eed , J. C . Tum or su ppresso r p53 is a d irec ttransc rip tiona l ac tiva to r o f the h um anbo x g ene . C ell, 80 : 293 -299 , 19 95 .15 . Y in , C ., K nudso n , C . M ., K o rsm ey er, S . J., and D yke , T . V .Ba xsuppresses tum origenesis an d stim ula tes apop tos is in vivo . Nature(Lond .) , 385 : 6 3 7 - 6 4 0 , 1997 .16 . A lnem ri, S . E ., F ern and es-A ln em ri, T . , and L itw ack , G . C lo n in gand ex press ion of four nov el iso fo rm s of hum an in te rleu k in - 1 3 c o nv e rt -ing en zym e w ith d iffe ren t apop to t ic act iv it ies . J. B io l . C h em .,270:4 3 1 2 - 43 1 7 , 1 9 9 5 .17 . C erre tti, D . P ., H o l!iingsw o rth , L . T ., K oz losky , C . J., V a len tine ,M . B ., Sh ap iro , D . N ., M o rris, S . W ., and N elso n , N . M olecu la rcharac te riza tion of the gene fo r hum an in te r!euk in -1 3 co nverting enzym e (IL 1BC ). G enom ics, 20 : 468-4 73 , 19 94 .18 . W HO . H isto lo g ic al T yping o f In tes tina l T um ors, 2 nd ed ., pp . 233 . H eide lberg , G erm any : S pringer-V erlag , 19 89 .19 . W HO . H is to log ica l T yp ing o f F em ale G en ita l T rac t T um ors, 2ned ., p p . 13 -18 . H eide lberg , G erm any : S pring er-V erlag , 1 994 .2 0 . W H O . H is to lo g ical T yp in g of O eso phagea l and G as tric T um ors,2nd ed ., pp . 2 0-2 6 . H eid elbe rg , G erm any: S prin ge r-V erla g , 19 90 .21 . Lauren , P . T he tw o h is to log ica l m ain types of gastric carcinom a,d iffuse and so-ca lled in tes tina l-type carc inom a. A n attem pt a t a h is to -clinical class ific atio n . A cta Pa tho l. M icro b io l. Sc and .,64 : 31-49 , 1 965 .22 . Japan P an creas S oc iety . C lassifica tion of P ancrea tic C arc inom a, 1sEng lish ed . Tokyo : K an eha ra & C o., 199 6 .23 . In te rna tiona l F ed eration of G yneco log is ts and O b ste tricians . F IG Os tag es: 1 98 8 rev isio n . G yn eco l. O nco l.,35 : 125 -12 7 , 1 98 9 .24 . Jap an ese R esearch S oc ie ty fo r G astric C ancer. T h e g enera l ru les fogastric can cer s tu dy in su rg ery and pa tho logy . Jpn . J. S urg .,11 : 127-1 39 , 1 98 1 .25 . Fukush ige , S ., W aldm an , F . M ., K im u ra , M ., A be , T ., Furukaw a , T .S unam ura, M ., K obari, M ., and H on . A . F requen t g ain of copy num bero n the long arm of chrom osom e 20 in hum an pancrea tic adenocarc i-n om a. G en es C hrom osom es C ancer, 19 : 161-169 , 1 997 .26 . S tran d , M ., P ro l la , T . A ., L iskay , R . M ., a nd Pe tes , T . D . D e stab i-liza tion o f trac ts o f sim ple repe titiv e DN A in yeas t by m uta tions a ffec t-ing D N A m ism atch repa ir. N a tu re (L ond .),365: 274-2 76 , 1 993 .27 . B rady , H . J . M ., S a lom ons , G . S ., B obe ld ijk , R . C ., an d B ernsA . J. M . T ce lls from bax-a transgen ic m ice show acce le ra ted apop tos isin response to s tim uli bu t do n o t show res to red D N A dam age-inducedce ll d eath in the ab sence ofp53 . E M BO J., 15 : 1221-12 30 , 1 996 .


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