Embed Size (px)
Citation preview
_r...&<'O l.alllle-.llllJ1ll) 11_ ..... "'.· •....... OO<lF __ !>'IV, \" ~ 0 C _,OG Hod.-lD.,.....C '91.'! SCOPE
Cellular and Molecular Mechanisms ofTumour Promotion and their Implicationsfor Risk Assessment
HIroshi YamasakI and I Bernard Wemstein
ABSTRACT
The majontj' of ColICo" lhal OCC\1r in h~m.nl and ,n ...penmenl.1 an'malsprobably ..,~\l from lhe ,ntor""t;On of tarld ",Ils ..."h many raclors. bolhmd0F"0~••nd nOFno.....nd thro~&h5.·...1111 prOC\'SSn, R<'C¢nt S1~d"" On lhelCIion of IUJI>Ollr pron>ot." ,~U"" ,hat they ,nl.ract "lib cell' bJ' mochanblnSdifferenl from lllosc of lhe: 'tulial1"1 <:amn""",' Phorbol est.... I~mour
prornoIon for namplo bond '" sp<afIc mnnbfa.....soociaIN receptors nuhtrlhan 10 ",Ulllar D:-;A nu. appears '" Ind to .lt011llJol;lllO In cdI surfaoo~ mhl uon of ",,.roolIubt commumc:aUlla .Dd modulauoa of «IIdifJerentlauon, TboK 11 .Iso ~-.dmo< lMI K>'tfall~'PO>of h"","" Q.I>Cn raultfrom • mullJ>.tql Jlf'OC'O'S .. ,th Sl."P' nalopMlS 10 lllosc of lnlll&tioo UIdpromotlOll Tbo apown: of bumans 10 ""''"',, promoters Iud .ano<lScoCaetonl ""'~ Ibtrd"on: pial ... tmporu.tll roIo '0 alrocuol tho 'llcidnloo ofsporilic b""",n <:an«n, Thlll. In a-.., lho <:an«r ",,1 m bwnana from""",""ure 10 "roo.....".l...,...... "dimponant lhal .... de>dop tobablo "olI~"
for ouch a ls h 11 .nlot:lpaud lMI f~nhtr SludlOli on lho mochan''''' of lCloonof l~mour prOlnOlm .1 the: ",1l~1a,:and booclw:mocal "",...1, ""U prc....odo .......approadte' for idonl1fjlnl po'ont;allUmo~. promcll." and for more "'''''lUIapproach... 10 mk ""mal;On ,n h~m.nl_
I I;'\"TRODUCTION
The: mulli..op onl,n of mOSI mah...... nt IUmoun IS probablj' due 10 lhe:,ntor"""OIl of ta...t «lis ",'uh mulliplc: r""lora. bolh endol"lIoU$:and n.,..nousTbne rompln. Upecll orcatcl~mU$llx l.ken '"10 aerounl "'bon oneall=pu 10 an:aI)"W q\llJll1tall,..I~' Ihc: cancer nsk '" "~I for spoaflceo'lronmmW cbmualls. Thus. 1115 unbbly lhal • lIDak bactonallllU!aJe1>aulnt. or otlotr lbon...rm lnII. ..-ill aa:ur:alely pm1IC'Il~ an:>D<II""'t'ly of
OS,
IS6 MnhoJ' jor Estimating Rut oj ChrmlCal Inj~ry
chemicals. especially if we art al.o concerned uith Ihe important queslions 1lf'datiw po'.ne; and 'bk a>S<:SSm(nt m humans, Furthermore. Slnc. th.", isinc,easmg .,id.nce that tumour prOmot.rs and certain oth.r modifi.rs a.t oncells 'hrough mechan"ms d,ff.,.m from lh= of cla"ical carcinog.ns. i' is.""ntial '0 onclude on Cance, rISk aSsesSmenl a>Sllys for 'umOll' p,omoters andolhe, cofactors
In g.neraL lh. 'aM'I}' of ollr appr",,-ch« '0 nsk asseSSmenl is htghlydepend.nt on our und.manding of basic mechan"m, of "ariou, >lag.s ofca",inogenOSIS. In thb pape'. we shall'herefore review promOlion and anempt."he,e'er possibl., '0 relat. Ihi' knowl.dge to the more prac'ical problem' ofa="lng cancer risk in human"
2 T\\ O-STAGE :\IODEl OF CARCINOGE"ESIS
Among ,'anouS experimental models of mulmtage cafClnogene"" mouse skintwo-stag. carcinog.ne,i, i. ,h. d.ar." example in which at l.ast Iwo disti""t"age" 'Iniliatlon' and 'promotion', are well defin.d (Berenblum, 1975). Thi'model. 'ho"'o schematically In FIgure L tnd,a""" se,e,al fundam(ntal properti.s of 'tn,t,ators' and 'promol'rs', A ,ingle application of a low dose of an'inlliator' 0' repeated applicauon, of a 'promotor' alon. do no, induce 'umours,le, 'umours appear when th. 'promoter' i. applted repeatedly aft., a 'ingkapplicatIon ofan 'm"ia'o". Th" indica'.s that two apparently non-earcinogenicagent> can induce turnouTS when appli.d toge'he' in the p'oper order. Sonc.during th.ir lifetime human. a,e exposed to a 'ari.ty of environm.ntal factors,and oft.n at low do..... it i, conc"vabl. ,hal "m,lar s)'nergtsllc in,eracllons occu,in the causallon of many human cancers, Thus the cancer 'risk' for a gi'ensubstaoce i' highly d.pend.nl on what other .ubslance(.) ,h. Ind,Yldual tS.~posed 10.
It i' also .,.'id.nt from Figure I thaI the a<"ion of an iniuator IS essenliallyirm'er$lble. When an ,ntt,ator, for ."ampl•. 7,n-<limelhylbenz{ajanthracen.(DMBA) i,palnted a,a ,ingl.do... on mou... skin, the .xposed mou... Can I.ad anormallif. and not dndop skm cancer. Yet ,fas long a, one year la"r the mouse" exposed 10 repeated do...s of a lumour promoler, lumou" "ill appear(Boulwell, 1964), Thus. th.re ""S" a long_term 'memo,y' of the action of aomlllalo, on mOU'" 'kin. In on....n.... Ih••UbIO<lU.Ol e~po,ure 10 lumourpromolers determine. whether tumours appear or nol. 11 i' importaOlIO m ....thi' point ,n ,i,. asse<Sm.nt for "a"ou, en"ironmental chemical', 'ince ifIni,iato" are UblqULtous ,n our .n"ronm.n, th.n lhe e".01 of '~p"'ure 10tumour promoters could delermin. the mcid.nce of speelfic human tumou".
In conlea" to 'he ",,"ersible action of lhe initiators. lhe aClion of promoters.al I.ast during lh. early Slage' of carcinogenesi,. appears to be m"r$lbk(FIgure I). For oxample. when !he prolonged applicatIon of a promoter i'disconlinued. some of the papilloma, that ha". already appeared on mouse skm
Cellwlar ond Molet:lilor Muhonisms oj Tumour Promo/;on 1S7
, Tu"""'''
, • ..,,,"". Tu"""'''
, • No 'U"""'r<
• --, No 'u"","r<
, • - Tu"",""
• PO' ""_, No ,"moo"
, • - No ,"""'" ..
• • , " , No 'umeu"
• • - Tu","""
:x., 1I.""'.ea'''''' of 10"'''''' (h>o" dOM)" 11."""",,,,, of ionia!'" (Jew d_)", 11.""1"."",, <II "'0""""
Filur< 1 Sohemaliocd1Olr.mofmuhi.la~mou...km .....nolO....;.
may aCluall)' ,cg,es.s. Th,. diff.",nc. ",'ilh =p""110 re,'e",bihl1 rna) he duo 10'he factlhat ,n,lialors or lh.ir m..abohte, gen..ally bind co"ale",ly '0 cellularDNA. RNA and p'Ote"'. and produce heritabl. chang". ",h.rea<lh••ction oftumour promo1<" ap",a" 10 he "a ep.gen..ic mochani,m,. and 'Iley are muallyno' mUlag.nic (We",,,.in,, 01..1977. 1979b. and lee helow1.
Unu] ,,,,.n,ll'. on. could ""ondor ",h<1h'T lh. mod.1 of '",,>-s'ag. carcin<>g.n..i, apph.d only 10 mOu" ,k,n and ",'as nol a g.ncral p,inciple '" carcin<>g.n.Sls. Ho""",'or, lh.", arc now ,",'cr.l o'ller """,,m.ntal mod.l, of lwo-51agocarcinogen",s (Table I). Th"o i,. fOf examplo. accumula'ing ""donee lhal h"orcarcinogen",i, in ralS occurs by a muhi<l"P p=" ",'ilh initialion andpromouon \lagos similar 10 lhose scen on mouse ,km (P..a,no 0' 01.. 1977;Shinoluka e/ 01.. 1979; farher and Cam.ron. 1980; P;'ot fI a/.. 1980),
Th. agenlS that act a, tumour promo"" in "anou. u""os d..play conSlderabl. chemical diveroily (Tabl. 1), Thcy includ. phorbol eSl.", phenob.""",hann. ph.noMrbilo], DDT, .Ic, Much of our ,ccen1 knowledge .boUl lhemechan;,m ofaction of lumOUT promote" ha' com. from "ud,,,, of lh. phOTbolos1<rs .nd lh.i, dcriualiu", (Weinstein 0/ al" 1977. 1979.; Slaga n "I" 1978;Blumherg. 1990). Th. mochan..m of acl,on of the non'pho,bol .stor lumOUTpromoters;, much 1." ""ell undo"lOod and it seems hhly ,hat nat all oflh.m actvia lhe sam. m«hanism! a\ phorbol eSl"', Ho",',,'''. once th.i, action i' fullyund''''ood. " " po\S,bl. lha' they will all 'hare some common final mochan"mThu,. only ,ocenlly ha. il become apparcn' 'hal DNA,s a common and critiealta'get for a varicty of diverse ;nt\iatars i~dudi~g ladiallO~. nttrosamlnOS,polycyclic arom.lie b)'drocaroon,. a<omatte amin... vin,'l chloride. etc, 11 is of
lS8 M"IrtX4 fOT UIUMIUt1I RuJ< of CMmlCal In)""
Tiuuc Ini,ia'or P,omo",I'NJ<b<>I ..,.....Iarnl u"""""
SkLn Mou.. Poly<oy<lic h~<!'(>C,I.,bon. Cro'on 00.1. phorbol 0<""Ml'o"NG. '.propjoW:to.... t.I"""Klrnuretha..
~." MOll.. OMSA '",." 1010... OMSA '"SlOlnll,hStomach ." MNNG Croton 0,1u,'.r Mou.. DMN ~,~
Lun, Mo... DMN PhorbolIV",,·pllo<b<>J u_~.
Skrn Mo... Pol~~I" h)"drocorbon< "'nthralin, iodoo<:eUl,..'''«TIS, cit"'. orL .urface-acti'~ a..n"
L"'.r ." ...... F. DEN. T.me'hll·.'·,S· Phenobarbital, DDT, SHT.dirnethyl4-aminoalobcn- TCDD=,
Mou.. DEN K'Lun. Mou.. Uretho"" '"'o~ ." MNNO Bile acrd..,- ." MNU C~lom.ol<. sarxhann, normal
"oMMammal) ", ~.u"on. }'-radLO"on Pro!oelJn
"'MTh~'roK! ." N ·blJ (2-hy<! ,o'l'prop,1) 3...m,no-l,2.4-'nuole
ni"""'fIl1'"In..." ... ." I.2·Di""'thl'lhl<!r:Wll< Sodium horbi,u"",,
So< Slop " iii, 119'*' H..k" .... (191)) .t>d 0_,,"_ If>ol W"""". (1911) for ir>ol.......1mo<........
ont....., 'hal ' ....0 n..... , rttently d.",n'oed daSSC$ of luonout promot.... on mouse.kin. 'M illdolo alhlo'd ..Ieocidin alld ,he polyacell'" apl~..ia'oxin. appear to
acI b)" mecha."ms "'1)' similar 10 'ha' of phorool ...,.... d...p". ,!>e" qu".diff.~nl chemICal S1ruc'ur. (Fujiki tI 0/.. 1981.1982; Sugimura.1 al" 1982).
3 MECHA:'>'IS:'>I or ACTION or TUMOUR·PROMOTII"GPHORBOL ESTERS
hiS nol our aJm to ..,,,,,, all oflh. kno....n .ff",tS of'he phorOOI ........ On cells(1M number of .uch effect. is enormous). bUI ..... "..ould lik. 10 di"",.. comingene..1 'ypes of eff",,!. panj(,ula,I~' those lha' appear '0 be rel."ant to themechani.rn of tumour promotion. ThIS .ubje<:l ha' been ~.'ie....ed in d.tail in lhe
proc_hnp of ''''0 =::en, .)m",*," on 'umou, promolen (Slap~' tJJ., 1918;Hocte, ~I tJJ.. 1'1ll2).
).1 S,tcilt CdI.r•• KfC'tJIlO<S for ftoorMi £$I",
SIIIlbeo .,Ih odl "U!I= .).Iems hIl... mdJc:oted I.bI.I lhe pnma'1' K\JOIl of1l1lDOU'-proIDOWII pborboI eolm We< pAot .' clw: odI owf..... , Thus lheC1pmu... of <:db '0 pboobol n«n <::UI 0_ IDCI'eUed membrane 1l1lllhlY.ltIrn'U:d uptake of 2-dcoJl)c1lOCOOC u.l "P.nd "Rb·. mllibillOG of EGFbtDduIJ 10 <dlular n:«p.on.. 1)1IIITpWl' "'''ll'KUO'' .,ch ,......"111 fXlon.. .11erfd
cdl acIbn>oa and WlCOUphnaof~"""'P: ~on...nd u>:,ea-:!.lItD(n"Uofmembra... pl>o<pl>obpod>(o« ....'ml>«1JI~I •., 1971. 1979a1. lnemeraJ.lhm:nuu. ,oad corrdation bet..-en lhe 1_'''p",m,,"''1 KI1'") of phorboleoten OG lIIOUK <kin and !h(l,dJ~ OG llIIml~ OIructure and fWlCl>OG (La:and 1eIn. 1918; DroedFJ and Slumber" 1m y.m....h ~I •. 1981).
Ie P~>oWy b«:n l'O"ula'C'd lhlll lilt phorbol "'len may .....rp liltfullCllOfl of. cdl .urf..... rccq>I<>r.~ IIOnnIJ fUllC11Ot1 Ii 10 mediate lhe 1It\J0lIof. y..,,,,o-he ilknu/ied """"h It,ula'or or 1>onnono (La: and ....",,,,,,,,,n. 1978.W",nOUln " •.• 1979b). ConlJ"0111 ,,"h lhll h)1)olhes,. .roo(I) lhe 1.,.' conttntra,;onl., "h"'h ,he lumOu. promol~' 12.. Q ..wrtde<'anoyl
phofbol.13.acelal~ (TPA) and den...,,,... acl ,n cell cuhure (10- 0 10
ur'o ,.);(2) lhe ,~ma,kabl~ s,milarilY' in Ilrllctural .equi.~m~m. I«n "hen a "arlely' of
pho'bol ""~" and relale<! mac.ocychc d,'~.pen~. are \~S1e<! in di,..,>csy51~m"
(3) lh~ hiihly' pl~iolropic .nd ,.....~ ...,bl~ ~rr«:,. of lh~>c compound,; .nd(4) ,ndll"cl "',.j"",,o lhol lhe pUla,,,'e TPA nc.pIO•. 11k< ..voral OIM' ......p1"....
displ,,)s dO"'l1 regulatIon
Specdic '_pIOro for pho.boI ",te... ha,.., ,nd«<! bttn domonotrale<! ....c~1ullln crude membra... fraclionl 0' "n ,nl.&Cl cells u.,nc 'H·ph",bnl.. 12.13-d,bulyra'e ('H.PDBU) (fo.......'"". SlumM" ~I •.. 1982). Tht:rr uud," andl~ of "'hero hIl... >lM>9ol1lh1ll phorbol <Ste, rcceptor> ha,.., a hiP. affinny forphorbol nt<"l'$ (K" ,.....,. from 10 n.. 10 SO n... tkpmd,"I on lhc cells).nd .hal9dI cdl bas .boul 1-2" 10'...:b bmdlnl SUts. The bmdlnl of 'H·PDBU anM "'hib""" b)' '''no'''' lumout-promotl"l pborboI e>\<'l'$ ODd.hclr ,nhibuOf}abollly JI'Defally <:onrlal...,lb lhe" lumou,"promoI1DJ K\1';I) on mou.. .t....and •.,.h their bOnc:lltmical or boolopcal K'It"'Y e~ on culillfed odlI(DrwdFfand Bhunbefs, 198O;Sbo)...bandTodaro. 1980; BhimbetJ".... 1982;y.m....kI ~l aI•. 1982a.b). TheM~ hiah-affiml) pborboI elUf ~Ol'$
hIl,,., b«:1l dn..:ted in" ."nety ofculillfed cdIs and UHuts. .i\h clw: 6CCpOOG a/mslUl't ftd blood t'fIIJ (Shoyah tIId Todaro. 19S0: HOro'lm II III.. 1981:ShimbetJ "tJJ... 1982). Tbc m:ml find,np !hIll pborbol ..ler b1ndlnls"eo abo
160 M.,Mds/ct EslimJirinil JWJ: o/Ckm.,,,, InjUry
emt mloVlCT ammalj, SUCh as nematodes (Ouuana tl ai, 1981). hydra. and,pong., (unpublished oh..,......t'on.) sunes, a h,gh d.gree of phylog.n.'icconse:r"alion of th= receP'ors.
These: findings sun'" 'ha' such recepto.... are norttl#lly U$cl by ce-rtainendoFflous agon .." or lipnd._ Se".ral all.mp,s ha>" alr.ad)' been made '0iden,ify Ih. pu'a'i". physiologteal Iip-nds from body ftulds Or t'ssue U'rIC'IS bUIIhus far ,"'lthou. s=ss (f-{oro,.,tz ., 0.1.. 1981. 1982a; Blumberg" 01" 1982), Ifsuch substances do .xi't ,hey could oxen b'ochemical and b'olog>cal .ffeoo1S.,m,lar to those: of phorbol e<ten and 'hus CQuld play an ,mportan. rol. a,endogenous ,umour promot..... or a, modifi..... ofcarcinogen.,i,. It '. imponanl'0 no" 'ha' TPA and related CQmpounds are po..nt modulalo.... of "ariou,pro8rammes ofcell diff.rentiation (W.iMt';n 'UM.. 1979b: D,amond.r 01.. 1980:Yamasak,. 1980; Yamasaki., oJ.. 1982a). "is CQ"""i"abl••ha. tlte pUlati,..endo(l<"nous Iipnd for phorbol esl.r recep'o.... may be a f..,OT (or f..tOn) ",'hiehcontrols nOmlal cell diff.rentialion. prolif.ra'ion and <!e,·.lopmen!. Recentstudie, indicate that de<Jl1" their d,ff.rent .h."""al structures. '''0 n..... clanes oflumour promot.rs. teleocidm an<! aply,ia,oxin (F ujiki "01.. 1981, 1982), bind to,h. same cellular recepto.... as the phorhol ester 'UmOur promoters (Umaa""a .'01." 1981. Horo"'itz tI 01.. 1982a.b). Thus ,h. acIion oftbese: recepto.... may nol beconfined '0 the phorbol esters and could ext.nd to a brOl'd range of ,..Illurallyoccurring compounds, On the o'her hand. not all cla.... of tumour promote....acI ,hrough Ihis receptor ,y".m. Th••ff....s of tumour promot..... on celldiff.reotiation and th. possible rel."a""" of these: .ffec" to the moehaoism oftumour promotion are di5Cussed belo,., (3.3),
3.2 M.mbran. Elf..,,, ud C.II-Cell Communkatloa
The binding of phorbol.'t..... 10 lheir cellular receptors i' followed by a numberof membrane-rela'ed .lfeoo15 (for revi....·.... W.in".in or oJ" 19791; Blumberg.1980), Among the...ffeclS. the recent finding that tumour-promo1lng pborholesters inhibil 0011--<:011 communication may pro"id. an imponanl clue 10 theprocess oftumour prom01ion. 'i""" cel1--<:e1l communication is thou"'t to ptay acrucial roll' in the control ofcell prolrf.ration and differentra'ion (Loewen,teln.1981),
Evidence that ,umour promol..... can ;nh'bt cell---<ell communication "'as firstind.pend.ntly obtained 1»' Youi ~I al. (1979) and Murray and Fitzje"rald (1979).These: in'·..tipto.... found lhal TPA and related phorbol ..ter tumour promo......inhibi' metabolie cooperation bel,.·..,n cello in cullur•. Al,hougb the .lfoel' "'·.restriking. th. rnctabolic cooperahon ,eoohn,que for the stu,l}' of ..11--<:011 com·munication ha' ,,'·.rallim;tations:
(I) Smc. m"abolic cooperalion j, the 'ransf.r be''''..,n cells of relali".ly largemole<:ul... tb. absence of thi' proces' i' not ,n it..lf a demonSlrat,on ofcomplete communication "lCompet.nce (Lo.w.nstein. 1979);
CdlulaT and Mal~<:ular MHlltmL<m.f 1)/ TUmellT PTDmOli/Ht 161
(2) Imlabol~ ~oo~ralion i' a relalivel)' complex process in"ol"ing ...veralbiochem~al reactiono. and
(3) lit<: ,nhlb"ion of melabolic coo~ration may reflect inhibition of previou.l)'c<labh.hc<I and Or <k noel) formallon of palhway, ofcellular commun;calionin gro"'ing cuhur~,. and lhe procedure use<! don nol di'tlnguish Ihedifference,
An a~<ay lhal o,'oreomes Ih.... limilation< i' 10 pa" pul..., of electric currenlIhrough intraccllular m'crocle<:"odes from lhe InlCrio, of a ccll 10 an e'lemalmedIum and to moa,ure lhe cor....ponding electrical polenlial in thi' cell and in aneighbouring cell (Loewen'tetn. 1979), B)' mean. of ~lICh a melhod. il hasreccntl)' been demOnltraled that tumour·promOllng pho'bol eslen re'>'01"l,blyinhibil both lhe formalion and maimena""" of electncal coupling of ~ulll:red
human epilhehai cell. (Figure 2). Tumour-promoting phorbol eSlers did notchange the membrane potential. membrane resi~lance.or ,ro"'th rale ofthe celli.'Uuesling that IUmOur promotors <:an ,nhib,t ce1l---«1l communicalion withoutaffect,ng Ihe general pro~rti.. of lhe ,urface memhrane, Ho...·"..r. il i.importamlo emphas,u lhallumour prOmOICl"l may inhibit cell---«ll ~ommun,·calion Ihrough indirect eflects on membrane structure or function .ince lhe)' arecapable of o.oning a "ariot~ of luch orre<:lI. Among ,'arioul phorbol eslers and
o
I~ •
0
•
t.~
•0 - •
"'"
FiJU'" 2 Elf0<1 ohddll'on....mo"ol ond ...odd,loOll of 100 nl ml12-Q.'<tf1dcconoyl pborbo!.lj·occ",,. (TPA) on <oupl,nl bel.....n Fl colis. Am'.... indiat. times of addition or mno,,,1 ofTl'A. Tl'A " ......~bycar<funy...oh,nalbe<dI1l.)~j"........itb eul1U... rD<dium <OIItaintna 10', calr O<<W<I. 0, ""nao!.•.TPA lrelted ctll1 Rtpl'(l(luctd .."Ii ptrm,\Uon from Eool\'l(llo"<>I. (1981)
162 M.tltods JOT UlimolUlg R"'k cf Clttmkal InJ"'Y
r.lot«! compounds tellcd, there was a ~O«l COlKlation bct"'l:n tllcir abllit} 10Inh'bil intercellulor transfer of eleclrical cuff.n!. lumour-promollng aCllV"w.;and 1M ablhly 10 'nhlb" POBU blndlOg tn the cells (Enomo,o n 01.. 1981:Yamasaki .. al. 1983).
The faet lhat cena,n lumOUr promOters can compktely mhlbit electricalcoupling of cell. implies lhal lbey m''''t 10lally inh'bil ,,,rious types of ",,11---<:e1lcommunica"on, n"s block in interttllulor communicalion mIght in a compkxti ..ue produce disturbance in prohferation and cell differenualion. and Ihusenhance ,umoUr fonmuion, In !'iro upo"menlS d...:ribed by Raid (1913) arecon,,,lenl "'ith this hypo'hesis, He examined by electron microscopomorphological changel in mouse skin expo$ed '0 TPA and found ,hat TPAappo.red to induce tbe .ppoarance of intereellulor ,~s bet"'cen epidermalcells. Thcs<- cbangelcould be as.sociated "ilh tbe los. ofcell---<:ell commumcation.The same: aUlhor also reported lhat TPA ,mpaired epidermal cell differentialion.Ho,,'evef. "hen ethylphenylpropiola'e. a h)'porplasmiogemc agent "i,houtlumour'promoung .cti"ity, ",a, used there "'a, no "'idening of tbe intercellulorspaces and II>< Inhlbllion of cell differ.ntiation ",a. not obse ....-.d (Ra",k andBurdzy, 1973), These in .-1..., results are consiStent "ith the hy-potl><sis that'umour promotion i. reloled to aberrant ",,1I---<:e11 communication and aberranlcell differontiation (see 3.3 and 3,4). aI, hough oth.r "planations ha". nol b«nexcluded.
Mort r""ent !ludies hav. shown 'hal me:tabolic .ooporauon " also lOhlbtt«!by a "ariely of non-phorbollumour promote.. (Williams t1 01" 1981: Trosko.101.. 1982), On the o'her hand other inv..tiptors ha"e found that no' all tumourpromo.." Inhlb" melabolic cooperalion (Umeda" al., 1980: Kinsello, 1982).The possible use of 'hi' phenomenon for screening of tumour promo'e.. i,discussed in ~llon S
S,nce it has lonl! b«n postula'ed lhat aberrant differentiation is invol"ed in theprocess of carcinoll.n..... tbe modulauon of a ',,"ely of programmes of celld,llhenllalion by tumour promo.." has dra",n considerable auention(Wein"ein tf 01.. 1979a: D'amond tf 01.• 1980: Yamasaki. 1980), The phorbolestor tumOUr promo,er-< can ,nh,btt diff.rtnt,at,on of many types of ""n. bUI ilshould be empha...ed 'hat w;tb cenain celltyp'" 'ht)' enhance differentiation(Tabk 2). At first ""'I, Ihest dual effecti oftumour promote.. appoa' confu,inl!.On the other hand. ,hey rna)' ..plain ceram " ..utspecific eff""". We beheve tba'Ihe ",hibitory effect is direcl1y reloted to tumour promollon, We presume that inthose tissue. In wh"'h pborbol esle" Induce lennlnal d,ffe..,nllalion_ Ib.y do notacI a. tumour promote.. , The latter effec' rna)' partially explain why some: ofthese compound. haH an an"l<uka.mlC acUOn (Kupehan "al.. 1976), In IhosetiMu" in "hich term",al differenllallon IS ;nhibl'ed, there "'ould be a rapId
C~lIular aN! MoIrn</ar M",,!rortUms of T""",u, P,""""liM 163
accumulallon of und,lTettnllated ""U., ,,'hieh ""uld ra'our the outll'o,,~h of theiniliated ""n.(Wein,tein <I aI., 19790; 't' amasaki, 1980). A similar bypoth..i. "a.ptt..nled by il<renblum (1954) from ear)ie, 'tudi<s on mOU" ,kin. Figure 3il1ustra,<s his v'e.... of lho medu.nism oft ..<Klal!" mou.. skin camnollen..i.; "'etonsidcr lbal lhe ....ulu obtained in duo strongly ,uppon ,I", hrfJOlhes>s(Yamasaki tI al.. 19821.). A ..mila, hrfJOI!>esis has Jx<,n proposed 10 explain thoorilin of hored"ary tt1,noblastoma, in po....,n, "hose llenelie predisposi,ion (.mutalion) i. ""n'idered 10 be responsible for initiation. al\d. radOr controUinllthe diff.,enlla,ion of 'ct,noblaslS lui' lxtn po<lulated to playa rol< in thepromolion stage (Matsunap. 1979; SO< also section 4),
The inhibition of ",,11 dilTe..nlialion by ,umour promoter< i' nol du< to 'OX'C
Tabk 2 Modu"',,,," of cell ditTe=>lIallon by llImour-l"0mol,n, phorbol "'n>.RtprodllCCd b) pcrnussion of R"'tn " ...... 1'1..... York. from 't'ama..k, 'fI>1, in Hed<1' <I01.• Co,,_,u CIKQT<_siJ 4IId lJi<JJcfklll Effttl> of T"""" p,OIfft}/m. 1912
A, fnAib<,_ o. <kla}' of diff<m",al;or,Mum'" ef)throl<uka<mlX (Fn<od)Muri". 3TJ fibrobla't>"Mun". neuroblastomaMum ".ta"Mun". I.nom.o"Mun". ml~1o'od Ioohem,.'
Mum.. ClH lOTI 2 cell.Mou.. epld<nnal cultu,..ChlCk embf)'o myobl.mChick crnbf)'o thond,ohLo..uC1l>ck crnbr)-o """'''''Ktnic <-cll.Rat mammaf)' cxrc,,,,,,,,aHa""'<f eptd<nnal cullumNemalode (c. ",garr.slS..urchtn
II. loJ.M:li"" '" ".",.10'"'" of dif!<""'OOIIonMuri". e-ryrh'oIcukacmlX (Rausbe,)Mun". mrelOO<l "ubem..'Ch"k m)-.IOO<I Ieun""""Hum.n m)-.loo<IlcukacmiaHumao melooo""Hwnao T I)'mphobl.".
Hacm<>JIoblo ')'O'hcsul'PId oo:umulat,onNelln.. fOnnal.,nNelln'. fonnationM,lan'lI .ynlhc<i.MorplIoIOCl, phaJo<lllC
",ivityM0'l'holoJ)'K.ralinWtI"'nM)'Olube fonnat>OtlSulpha'<d 1"0I<oJI),cxn .ynthtsi<Molan,n ')'II'bndDome (onna,.,nKorallnlZXllonEarl)' dc'~Iopmc:n,Embf)'o,.".."
Homo,loblo'ln''''''i.MorpboloJll. pha,OC)''''''. e"'.MorpholOJll. pha,OC)~osi.
Morpholol)' pha'OC)~o<is, ct.Delldri", formationE_",..u< formation
• Dd1'e_""t""';, del.,,«1.....' "'" ''''''I'I<ItI,-lN",.«I. h,- pho,boI <"'", Inhibol"'" n' obom<d '0"" "'If .....'" ~'U "sed ,n .,.ltu", m<>l",,,,. hooo"",,", TPA ,n<I-.dJlfmn".",,,, '" lho prnera of {etal calf serum (lr.o",k.b< ,,01.. '981).So< V......k, " M, '191.1.0) fa< dldi..;d",,1 m<t<O«<
INT'ATORleQ 9[.jP.OM9Al
I
~-<hI*>y"'ll _'"*,,,1"'" end"'1_'"1'''''
At>Qeoo 0 oce 01..1",led1"",,,,,,,,,1 ceh
/4li>eUiooce at oeIs..In -."'"GII......"" .....(I.......,.. 'l
FiJu,,) Sclltmal., diq,.m of. "",l.il»e Il>«'han"m 101 IWO-ltaJ< <hem",.1 "",.o;nOI'''''''"'' Mo,lIfIed with p<mli",on [,,,,,, !l<,.nbl..m(19Sol}, •• initiated ullO; i.J. ulll with d,vioi.....pao<it~; _ dlll'...n""IO" h~ dlviJion; _ dlll....nl;.IIO" by mO""....OI to ,he....rf_; S, ••t<m ull; hl. m •• m,. d. dill'.",n"'l;nJ «110; k. kcrallo
effectS, at>d 1M llIhIblUoa IS ..,....,..u) m.-.niblt afIn lftDD'-aJ oJ \he 0Dmp0Ilro;!
fTOfl> CIlII"'" me<!'lI\lI. For <:U.lIlpie. ""b COIlIUI_ aP"S"....,11'A """ llIhIboltermmal ddfetmWluoa oJ CIlIlllm oJ m\lflllt nyllitokubmua cdIs for loa...Ihu! yean ..,I.!I about 210 scnal po...... H~~.I.cdh .....\'r lew IbtttidcnUl) as nylbtoods ...,.,. &fie,- mtoseqllnll lftDD'-aJ or 11'A. I. cdlsdlII"ermWlu at>d Iooc l1Ioett prolIferauve 1'&I'K111 &fin ....-.nl odl dJ.__(Yamasaki ~t aI.. 1980, 198"'...a.. at>d UftPIlbltsblldn:tWU)."Theltl"lJll1llJddl"nmohalion or mu""" nytbroleukactma cdIs II prcaded by I. accurnilbUOlI ofC),opb>m>l: ""bon mll.NA (Mam at>d 1l.,1'Ir.11ld, 1911). As10.., ..11'A IS pmmlltl t:IlIlll.... medium. s10buI mRNA does not IKC'Umllblt in lbe C)'opIasm bul an"mno'-al of11'Aarap;.!8OC\1mulauoeoflloblemll.NAOCCIIIS (YamasakI ~, 1J1.•1977 and Ilnpubllshtd obst;n·alion). II ... IbemO..... C\'IdC1lt lui TPA does noldam.1i" but ,"pprcsscs lbe .xpr...ion ofs10bln ..nes.....hich i' wnsid.Tt<l 10 be •k~' evenl,n the lndllClion 01' .rylhroid diff....ntl.t,on. On lhe oth.r hand. c.lIsdi,'id••nd prollf.rat. in lbe presence orTPA.,. IImil.. ,al. aI in lh. wnlro!medium (Y.masaki tl .,J.• 1980). We an Ih fo.. o;onclude th.l TPA does nOl.ffeclille function' ormesl ofille ·ooll....k nl· FtIeS •.-hich a'" ne«sslry fornormal oeD prollf.rallOll bUI ..lea;,..,I) modullll"llIe expression of JOneIlhala.... in>oI\"«1 ltl eeU dllf.....nliation.
Al!boup lho 11'A..ndl>Ced modubtion of,me expressIOn iJ sdecuve, ''Inouspropammes of oeD dllf.......lialion 'n: all"ecIed. Tbe5e tDclIIde~l)1Iu..e-. '''Utnt fonnalJO:l>. hpod. ')1Iu..e-. aw\aJ>In ,)'ntht<rs.. mjOWlt t)1Itbe:s>sat>d proICO&lyc:an ')'IIu....... aD or ..-Iuch 1II\"OlYe Ihc Clljli "OIl ordllfermlljl'efof...,n. HO" CIJI. 11'A modula'" the Cllprasaotl oflhlS ..1& '-andy of ........i!boul .treallll_ bouoe-teepm.c fUllO:UC:WlS' Prauma~· 11'A lIIll\>eaCelaprnecs.s thaI is _ 10 a '-andj ofl'fOlrlll'll"l of differmWlUOD ()n,o
\l<>l'PbIt prom.s nuPI. I. c:dI-<dI _111'111'&1101'1 (.... 3-2 abo\-4 011 1Mother haDd, IoIDOI: 11'A """ IIIllllCDOO Ihc dttrermlJaUOD or hxma\Op<*UC andleubemlc edit that UtSl as 'lOpe oelk. other ......,haro..... mU<11l1<o bt lovoh-ed
3.4 Model of TPA Aellooo
Besides lhe effects described aoo,..,. TPA and ....I.,td lumour prOm<lters U,...number ofother blolos,ical and bioch.micaleffects (see Siallati aI" 1978; Heckern al" 1982). Amonll th.... Ihe ,yncrsiltic inle~lion of TPA Wilh viN"s to
.nhancinll oeillransformalion (Fi,her and W.inlleln. 1980) and modulalion ofimmllnololK'lll)'Slema (Keller. 1979) are of parlic:ular In"'rest wnh respe<;l 10the mechanllttl of Qrl:inoi"nesis.
FiJUre 4 i!luSll1lleS a poWble model of how TPA enhallCeS lhe oon....l'SIOll of""I"'ted oelk 1010 11lmour cdh. Tbe uuel1lCtion of 11'A .. ilb oellubt rectplOI'Iresults IJI lho allenllOll of a ''IC\C1)' of SIIrfaee membra... ptopnues. Thesemclu& allmld adll$on. altt:red uptake of fllltnents, allmld respome to JrO-ufaetOno, ah.md fUllCtioa oJ c:dI outface~ at>d IIIlpauul c:dI-all
166 Mothods f()l' Eslim/Jliltg Risk of Ch.""cal InjlAry
commumcatlOn. TMst ~mt; could mmu13le tilt OUlgroll,h arid mhiblt liltdifferenlialion or pre..,xi'tine iniliated cell•. Pre<umobly Initiated cells r",pondto tl>e lumour promoter preferentially Yu.pa and Morpn (198 I) ba'.., providedevidence tbat Initjallne carcinOllen, aller cellular oommitment to differentiationIf turnOUT promo'.r. act .pigeneticaUy. then it 1$ not clear how promotioneventually lead' 10 tl>e outarov.'lb ofauto nomou, Iumours. lbere " ",'idence lbaI
afle, protonsed ilO....'h of erylhroleuhem,a cells in 'he presence of TPA,differentiation i. inhibited by agents (,ucb as dimetbyl,ulpboxide or l>exameth.ylene bisacetam1de) that would normally acl as mducelS. ,uuestins that TPA
t.ltI.II •• 0:.."",," CO'"'1< "'1,0 'nul" I" •••b.,,,U,,,, ,"<e11.I.r c_I_OI to ~Iffe~"tlotlon ,...UtIO".i'.' ''''''"'/_"'. etc.)
._ _ .
1'PA OIMI"9, ....P'oro
1A'lor.~ <ell s.,I". ~'_t'tl•• ('"c,.ned ""Irl.",optI" ••Hero<! "'''Plo, f""ctlon. 1.~I.ttlon 01«l1_cell "C"'l.HI •• .,~ <_oj"tIOfl. etc.)
"<opo I"", 'i''''''~ c""tro1< .!Ml .It.,,,~ ••or... lonof c.-1_nl to dtff.","'I.ttOfl
t.~lbl'iO/l ofdl Ifore"t lotion
1
su"", .. ton of~I ff.~"11<t I""
FiJllrt 4 Cellula, n>odel of TPA aet10D
Cellula' anJ Moltcula, Muhan/sm.$ af T"",ou, P,omtJrion 167
can induce porman<nt changes In ditTettntlatlng cell 'y'tem, (Yama",ki eI al,.1990, 1981a), Similarly, Marks" al. (1978) ha,.. ,ugge>led that cells In TPA_lttaled mouse 'kin re,pond to a Gl<halone In a ditTerenl manner than thecontrol sbn <:elk These re,ull. ,uggest lhal ·lock-in· of TPA...tT""t may beachieved by changing cell response 10 a factor wh"h normall) conlrols <:ellditTerentia'ion, In addltion. TPA irreversibly Indu<:<' anchorage indepondentgrow'lh in adeno,iru, tran'formed rat cells (Fisher and Weimtein. 1980) and lncertain mouse epidermal cell lines (Colburn" al.. 1979), Further studies arerequ,red to determine whether lhese irr..'ersibl••tTee.. ofTPAinvoh,.cpi",ne\lcor g.netlc ..'ems,
4 EVIDENCE OF MULTISTAGE CARCI:>OOGE:-'ESIS ISHVMANS
The laten' ponod ofmany human cancers, i", the tim••Iapsed between ..posureto carcinogens and Ihe appo.ranc. of cancer. appears 10 be too long to beexplaIned by a "ngl. step event and is more consistent ,,'ith a mullistage process,Epidemiological studies have id.ntified at leas' 3<khemlcal" Lndu.trial proce.....or occupations lhat are cau",lly related to human cancer (Tabl. 3; lARC. 1982).
Clinical and .p'demiological data also prOVIde eVIdence that cerla,n humancancers are caused by the interaction of s..'.ral factors and a multi.lalle process,Table 4 ,ummanzes exampl.. of ""ch human can<:ers. Ahhough lhe availabledata do not provide dir«t .vid.nce ofa two-stage model $imilar to that seen Inexporimontal animal', the ..'idonce is ;n several cases con'istent with theinihatlon-promOllon model (Grttnebaum and Wern"ein. 1981)
The fact lhal th.... is a rapid decline in lung cancer ri'k in heO"y cigarettesmokers when 'hey give up .moking h.. been interpreted a. ovide""" ,hat,moking .nhances a lal. stag. as well as an early 'tage of the carcinogenic process(Peto. 1977: Doll, 1978). In facl. anImal 'tudi.. indicate Ihat ciprelle smokeCOntains nUm,fOUS subs'ance,. including both initiators and tUmOur promoters(Van Duuren., al.. 1971aj, Although the evid.nce i.I"" .xten,i"e than in thecase of 'cigarett••moking-lung cancer'. a .'mila, analysis indicat<$ tha'conjugated oeslr0llen. act at a late 'tag. of .ndom.trial carcinog.nesis inhumans; there" a ttlat"'e!y shOrt lag ponod bet",e... onSC! of postmenopausaloe'trogen use and occurrence of endom.trial cancer. and of reduction ofoestrogen preSCription and reduction in n.w Cale' of,ndorne'nalcanctr (Ji<k eI
al.. 19'N), There i. sugge'tive ",'id.nce that in cerlain .nd.mic regions of theworld phorbol ..,e" may playa rol. rn the aeliology ofhuman o«ophageal andna<opharyngcal Cancer. Th. high incidence of oesophageal cancer in Curacaoha' been aswciated wjth the use of a herbal tea made from the plant Crownfla"ens O,lorlon. I%8). Weber and Hecker (1978) ha,.. isolated a pho,bol esterfrom this planl and sug~led thaI this compound may pIal' a role 10 the disease.Nasopharyngeal cancer (NPC) '" As\a i' associated with Epstein-Barr (lOB)
168 Me/hods for B'imaling Ri.<k of Chemica/Injury
Table 3 t:!lllblilhed buman ctn:moeenic cbemal, and indumiJ.1 proctlS(IAI\C, 1982)
P"X'MUI. Auram"" <Nnufa<'u",2, Boot and >II"" <Nnuf"",u... and "'PO" (cen.. , ",posum '0 dum)J Fumi,u... manuf..,tu... (cenain ..po>u.... to dum)4, hop,opyla"'ohol manuf.."u... (monl-..,id ]>t00e><)S, Nickel ,<fin;n,6 Rubbe' maouf"",u"'1 indu"ry (cenalo occupotlO",)7 Und<"fOuod ha<ma'i', min"', (radoo)
Occup'"iMa/t:c;w><rtsI. 4-Ammohiph<n)'1 (a,omatic amino)2. A""",ic .rid ....mic compound.3. A,bdto.4. Bonze""S Iknzid'n< (ar""'-'-tic amll><)6. Bi.J(chlo,om<:'hyl)<'h<, (BCME) and '<o:hnical CMME7 Chromium and <hromium compound.8, Mu"ar<l p.9. 2_:";aphth)'lamln< (aromat" amll><)
10. Soots, lOr> and «nain mi""..l oii.II Vioyl chloride
la'rogo"ic ,xpc••mL Ana.l...". coo!O,n,nl ph,nacrtin2. Anthioptln<3. Cblo,amb""il4 Cblornapwl""5. CombIned chemolherapy ;""hidin, MOPP6. COfljugat«l """'0"""7. Cyclopho<pbamid<8. Di<thylitilboeottol9 Melphalan
10. M",bo..a.,o ..-"h UVA (PUVA)11 M)'.,,,,o12. Treo<ulphan
"irus, bu' £B VIflI. IOf«l;oo alooe appea" 10 be in.ufficient 10 e~plain lhe causeof NPC. Hirayanta and ho (1981) haH SUggeslro lhal phorbol este" conlained inEuphorblacea. ,,"ght serw a, lumour promote'" in NPC causation, b«au~ inSoulh Chma "-'here the incidence of NPC i' hlgb. ~icin", and nasal halm.ptqlared f'om th.... plan!> are e,"e..i"ely used. It is inte",,'IO' 10 nole Ihallumour-promoting phorbol es(.", .nhance in ";"0 replicalion of EB ~iru. (lurHau~n eI aJ" 1979) and Ihey alw enhan« in ,'ilro lran.formal;on of humanIymphoc)'t'" by EB ";fUS (Yamamolo and lUf HaustO, 1979).
The JlWllfaphlcal disuibu,ion of oewph.geal cance, in ChIna show, a veryhigh incidence to llOxlan County. Henan Province. The use of lobac.:o was
C~llula, and Moluwlor M~chani=s af Tumaur PromoUon 169
T.bl.4 Eumpl., ef mul"""i" and e, mullifacle, ca...inei'o..i' in human'
P",babl. P'ebabl.mltale, p",mclt., R.f....ncn
Lunloauc<r Ci.....u. ,mel:inl Ci.....l.. """kinl POle, 1917; Doll,19J8
Oe,.,phageal <o!\c<, , Phcrlx>t ",... W.ber and Hoohr.
'"'Ni"oo.am.",,' Picl:'ed '''Ftabl•• Chenl <r ala 1982(Reu,un', rod')
Na"'l'haI)'nl,,1 Ell vi"" Pherlx>1 ",.11 H,rayama and ho.ca""., +n-bulyr... m,lIu,l:iu', I)'mph- Ell vi"" M.I..ia inr<ct",o Dalldorf" aI.. 1964~
Endom<lrial """"'", ConJuptod
e'''''eF"' Jd"al,.t919eele" <>nc<r , Dl<tary rat R<dd)' <r ai, 1978
II...." CO""", Harm""". di<l'I)'. W.rn,,,,,n, 198J;,,, Day, 1982
Pr"'lal< <>""Of , , A....h 197J1I,41.ral HerodLlary , ),Ia"uoap. 1919""iuebla"emaL"er <anc<' Aftatexin II, Hepotiu, II un..nand P«...
1977Lunloao<,<r Cipf<110 ,mol:inl_ A'!><s'a' <apo'"" S<likolT and
Hammooo.1919O<:"'I'hageal. I.r_ Ci......" ,mok,nl + Alcolx>t <oo,ump.- TOj'n,,1979yn, and oral ,,""ity "an~~"
.",Iud«! a, a pos,ible a<lielogical f""lo' .i~ chick.ns in Ihis region also ha,'ean unusually hip incid.nce of oesophag.al c.ncer (Li n 01.. 1980). Epid.miological stud;~ hay. shown a d"ecl relahOnsh,p betw,,"n the incidence ofoesophage.l cancer, ;n belh haman••nd chic.en., and the consumplion ofplC.l<:d ,'.g.tables, Cheng" ai, (1982) have oblain<:d evidence lhal an exlract ofpic.1<d vegelables has promoling acti"I)' in llie mouse C3H lOT I 2 cell transformatoon ')'$tnn. The)' ha"e furthe, sugg<st<:d lhal dimeth}'lth;otelran;lrosodiiron.or Rou..in', ,<:d, whicb ....s isolat<:d from tbe pic.l<:d vogelable, may acl .. apromOler;n the causalion ofthis disease (Cheng ~I al" 1982), At the same lime, 'I.bould be .mpb..ized Ib.1 specific nilfosamines and mutagen' b",', also beenidenufi<:d in extraclS of pickl<:d ' ..gel.bles (Lo" ai, 1981),
On lhe ba.i. of geog,aphical ep,demiological 'tud,es and experiment.levidenct from animal ~tudies, ;e\"eral In\'esupton have lmrhc:m<l fat and otherdiet.ry racto.. in tbe pathogenesis of be""el cancer (for review, ... Reddy el at..
1978} Mllflnl JlOOItJ IDoW • rallld me In lbc iocidtoct ofcolon canc:rr IlIIOn,ind,,-.duals ... ho mlnu....wl '0 II.. Unued Sa.ln fTom Japo.n. t"""","y Of Pobnd(H......-I and K"nMra, 1968). An LDcrea.e in colon <ana. morub,) ralt ..-asobsm."Cd t'ftI '" 1M fifll. F""ra,ioo! of Im.InJ.I121lIS. Th ""'UOIImt1IW fanon.P<e5Umably dICtaT). COIltn1>ule '0 colon anc:er CIl ""1l. Fu.nllt. M~," an:required to <ktcrmtrIC to "'M' 0.,,,,,, "'" n:II«u tho """"fTe1lte of lnUla'on ...promocen ... boIlIlD 'M Amman din.
HWI:WI breut canotf .. an ompon.ull eumpk of lIl..hlMaft~:and one Ul ..Iudl WI"e'fIll pll)~ faclOn ....Y pia) a ..... ,wnout~ "T'Ione aspecu an: dllCllHed Ul deuJI by WtuISlC\ll (1981) and Day(1982). Willi mpe<I '0 PJO"'-"c cantn. In addiuoa to tho dueue IMI •~ chOlCllU) and IMI ....). hIl tho fill"""\. I!ICR is a Ialtlll form of tho<fueue ..-lucb " ofIm obwT"o'fd 0IlIy 1IIlCTOIOOpiCIl.' tho ImIC oIa"lops) anddoa DOt COIltnbu,. to mortably_ WbIk lito ..,.-adJ\lSwl mortab,y rale fnxnclinJClll lITOOlanc ClmllOal& ~'S dlSllDCI fIIl'lemS of ........p/t>c&l '-".na''''''(Sq, ., III.. 1%9). ,Ilt ~'&lc1>ceof 'M Ia,m, form of PJO"'-uc Clmnoma doano, '-aJY be,....... FOSJ1IphxaI n:p>DJ (Atazah. 1973). Thll JIlpnu !-bal 'bo'cndmcy fOI lalem P'OSLalt <ana. '0 de>'elop 1Il10diniClll Clmnoma II $'ronaJyInftllCl\CCd by cn'lronlllnllal fanon. Haensuland Kurihara (1968) Jhoo&'fd lhal'M morulily ralc of lITosla'. cancer &mO"i ,be IIrs' F""'!'lIuon of Japa~= ........ '0 H......" '$ J '0' limes "'abcr Ilian 'ha' of nau". Japa~.TIlil!'lip;.:! rUe ,n ri»: IUP!l'0TU the 'ole of eo.-iroomenlal factor'S, Since lMyprt:SuntObl~ acl 00 ,he lalt. Ila", of lh. d,'CI$C ,hey ma~ be mo•• Illlltla. '0'umour promole" ,lIan '0 ,"'Ila,o".
MalSunalla (1979) has propoocd a ,,,"'o-slall. mechamsm for ,h. causauon ofhilale.al h.redi'.ry '."noblaS'oma. Tli. In"i;"ion step is pr<:lumed '0 be due '0gene,>: predilpollUon. becaust bila'.ral r<:linohlas'oma cain u$uall~ $ho....au,osomal dom;nanl lnhe",ance AccordIng '0 his analysis of ,he alle..pccilk,,,,,,<It,,,,•••,. from 244 bola'....1 .aln of rctinob\a'loma. ,he•• I, I blpin'radass <:<>rn:lauon be''''cen 1M a", of pI,;'n" ., 'bo I"".. of d,aanosls of 'bod~ io bo'b tM ~l and left cya. Tbn resul, UI_ .plOlllhe ,WO>-lIlUla\lOntheory of ,!us d...._ propowd by K"udso" (1971) in .. 11..11 CIIK """ mlp, nocc~pect 'M OOCII~of botll ,be ~, and left .')'e Cllncer'S ""h,,, a lllon UII'k'"'terva!. II""' _uc cell mULaUO" IS a random and ran: .."",. He ,ulII"$wl!hal ;1 "-as mon: ....._ .... '0 assume lila, lito '<>.prnooo" of tnlllOblaMOmll
F'!C" "-as IarJdl dctcnDlJlCd by bolt flClOllO <;OIlUnOI\ to botb C)'C1. Mauunap(1919) bcl....-'fd !-ballbc promouon 1ta,,1Il bib'..... rft%lIObla5toma "-as a"~iD tho pro<:eU of <hIl''''''''lJlIuon.
In T..... a, .." ha,." bRed mgh,pIc fanon tbat ha.., boca omplGwl m litoCIl.....'ioa 01 other fOfml of b........ CIIIOCl", FunIICr "lIdJn arc reqwed to
<ictenDmc ..bd1 of thne actual!)' an .. ""ualOn or promoten or ' .. otbcrmedt'n_ W. mu>I aUo empllHlx !ha' ....... of thao: fanon may lUIIp!y anS),..,-plJCIlJl) .. ""Ilalon.
C~lIulaT and .'>1aluular MuAonism. of Tumour Promo/ian 171
5 PROSPECTS FOR SHORT_TER:,>t ASSAYS FOR TUMOURPRO.\IOTERS
At the p",..nt time. t~ most reliable te,t for the detection ofcar<inogenic agentsi. I~ rodenl car<inogene.i. a.... y Ho,,'C"er. more than 4 million c~mical,
are known and about 1000 new c~m;e"l••nter our en,-ironment ea<:h y.ar(FishbeIn. 1977); II C"'t o"er S400 000 and lakes at loast t...o years to I.'t a ,inglecompound in rodenl a....y•. Thus, II ISe><ential to de,·.lop more rapid shon-termlem as preliminary sc=n, for polenllal carl';nog<nS, EncouragIng progre.. IndevelopIng a bau.ry of such t"'lS for predic1ing potential <arcinogen! and forapplkation 10 human can<er nsk aMeS,ment has betn mad. (see Holl"ein el 01..1979; IARC. 1980: de Serre' and A,hby. 1981).
It i' w.ll feCOptud that m"'t curreml)' a'·ailabl. short·,.rm tCSts are baoed 00
the fa<:' tha' ecrlllin carl'inogen. bond covalen,ly 10 DNA and damage it, and ,buscan be assa)-.d for tbeir effecls on mU<alion and chromosomal d.mage. A. " ..•mpha.ized earli.r, howeveT, lumour promoters and ecnain mher modifyingfactors do not appear 10 bind 10 DNA and.re nol mutag.nlc In conv'.ntionalassay .pt.m,. ThUs. ,he.. agen...scape d.lection by mosl currently uoed ,honterm a,say', It i••Iso important 10 emphasize that many lumour promol.rs.nd olher modIfying factors may nOI be d.lected when tes,ed .Ione in rodentassay 'ystem., 'ince the'" .g.nt,. by de1'imllon, all: nOI 'trong car<inogens byIhem",h't$.
Thus. il is e,,,,n,i.11O develop shon-t.nn assays for lumour promoters andSOlo. progress ha< already been made in 'hi' area, For .~ample. a .hort-t.rmassay for phorbol eSlers and related IUmollr promot.rs was d.v.loped using tbeab;lily oflllmour.promo'ing pborbol e"ers to induce Ibe adhe<ion 10 the cultur.dish of murine erythroleuka.mia cells whicb u<,,"lly grow in ,uspension(Yamasaki" al.. 1979; 1981), It is possibl. '0 assay quanlllatlVely 10-20compound< in 'his 'yotem "'ithin a few day., Aquantitati"e .Iudy has shown lhalthe,. is, 10 g.n.ral, good corr.lation bel"...n the ability oflhesc types of tumourpromot.r, to IndUCt: cell adhesion and to promote mou....kin carcinogene.i•.Allhough thi' type of "'say doc. nol delec, all elaS>eS oflumour promot..... Itdoes d.tecllh. alkaloid lumour promoter leleocidin ",hose ,truClure i. dill.remfrom thaI of phorbol eSt.rS (Sugimura el al" 1982), Thus assays for alleration< inecll adh"'ion may be part of a banery of t",," for 'umour promolers.
The a....}' for inhibilion ofmetaboli<: coopeTal;on (see 3.2) for the ",...nlOB oftumour promoters was suu"'ted by Tr05ko el al. (1980) based on the" owndemonSlrallon Ibal nOt only non_phorbol eslers but also certain non-phorbol.st.r tumour promote.., 'lICb as saccharin. w.n: po,ili". in Ihis assay_Subsequ.ntly, i' has been .hown thaI a vari,ly orkno...n and putative tumourpromOlerscan inhibIt metabohccooperalion (Umed. n 01.. 1980; Tr"'ko eI al"1982)_ Thmfore. tht! slll'tm to be S llrOmlSlng SCTtt1\lng test for both llhorboland non-phorbollype lumOUr promote". Certain precaution•. how.,... r. loU" be
I n M~lhoJ.s for D'imoli"fJ Rw< of Ch~",ic(J1 In)",y
la~en ",h~n 1M a~>ay i~ u>ed, II ii h~el}' that agent~1MI cause non-specific tOlie.ffects at the cdl surfac. rna)' give fal.. >'O'iti". results. In addition, certainknown tumour promote" or cofactorl are appar.ntly negati,'. in thi' type ofass.ay (K,nsella, 1982),
Two-'tag. ;" "1"0 Iran,formatlon hal been Mmonstrated with mous<fibroblaSt C3H lOTI 2 cell' (Mondal and Heiddbe'ller. 1976). rat .mbryofibrobla't' (Lasn. e, al.. 1974), Sy,ian ham'ter cell' (Poiley" al.. 1919) andBALB 3T3 cells (Hlfakawa n al.. 1982). Thus far Ibese sYSt.ms have been u5edmainly to study mechanisms of cell tran,formallon by initiato.. and promo,...and ha"e nol been .xten,iv.ly explored Or validated as ",r••ning tem for tumourpromOters, A variety' of tumour promote". such as phorool ..t.... Tween 60,iodoacetic acid, an'hraltn and saccharin. do promote tran,fonnahon of C3HlOTI 2 mouse .mbf)o fibroblaS!< (Heid.lberger and Mondal. 1982). On th.other hand, ",cehafln was reponed to be n.sati". as a promoter in the BALB3T3 ceUtransformation 'ystem (S,,'ak and Tu. 1990), Since th••ndpoin, in atransformation assay i' thought to be directly r.levant 10 caremog.n.sis (T,'o.1981). thi' type of approaCh 'hould be encouraged and furth.r d.veloped. Itmight also Mt.", a broad., rango of 'umour promot... than a,<.oy' for specificmembrane effeelS
Although w. have de"'rihed only a f.w .xample. ofpo"ibl. ,horH.rm te".for tumour promot.,.. ,h.re are ""'ny oth.r cell s)'st.m. in "hich on. can .how a800d cotrelallon be'ween in ";/'0 .ffects and tumour-promotini acti';ty;" eli.."thu. off.ring th. pos"b,ltty of de-'.loping oth.r types of shorHerm t"'tS fortumour promote" (Weinstein. 1980). W. would hketo emphasize. how.,'.r, thatat the present time non. of these assay, ha' been sufficiently valtdated 10 that ilcan be used routinely as a ",reening lest. Furth.rmore. a major difficulty Inal1emplln8to d.v,se a unlv.",,1 assay for tumour promoterl may be th. apparenlli..oe and specie, specificity of differen' 'ypes of tumour promot.,... Theseargum.ms .mphasiz. the importance of further "udi•• on th. mechanIsms ofac'ion of tUmOur promot." in ord.r to desIgn a reliabl. ,hon-t.rm I.st fortumour promot." on a more rahonal ba,i. (W.in.t.in, 1980). Anothe< difficulty" ,hat for many tumOur promot." there are f.... quanh1\\'i"e dose-responsedata in ••perim.mal animal', thus making it difficult 10 make a quanmahv.c<>mpari,<>n between ,.sults obtained in ,hort-t.rm test, and the ,.-hol. animalas<.oy (Yamasaki el al" 1991). Thi. i' a major limitauon in our approach tod.,'.lop short·t.rm t.m for tum<>ur promot." and more quantit..i,'. 'dOKr.,ponse· s,udies are required in animal sY't.m,.
6 IMPLICATIONS WITH RESPECT TO CANCER RISKASSESS:IoIE1'.'T
If we wish to Int.rpre, ~Ix>ra'oryassays for .nvironmental careinOi.no. lumourpromote" and various olh.r modifiers In t.rm, of nsk in humans, w. cannOt
·>'Oid 1bt p.-obkm ofquanlJUOI""Ulnlpo!allO<l oflbeoc l'eSIdts. Tk recopl\JOll
WI DWl) human t:aD:::en .... c:allStd b) • muhlJUF pr<lClOS5. as described '" 1tI>Ipapa. COl J d nlNy compbcates 1tI>I w.l. nus IS partICUlarly ItUt for lumourJlO'O'I'OIen_. as_pb..." .1:10\". I"",,,'''' ''''Y fno aaunal dauo DOl lumourpcga>OtCS for .. lu<h dl"c<U ba'" beeIIorlOdxd ""'" • ....x ~,e ofdo5a.. t.-s
"'" .,.ao.o...oflbo maJOr Ii5<>n pertmmtlo lb< problem ofnil '''es$ Wlll for IUlllOUf
promolcn IS lb< qlkSOOfl of Ih=bold or I>O-dfKt..so.. k'o-d. II .ppear'J lbal. '"COIll1tillO IllJlllllOrs. IWI"OCMIr promo1cn .... IDOf" bul) 10 ba'" :albrabold foretrec-tS(W~IWl tf orJ_. 1977; Boul.....u tl orJ. 1m). To ou, knov.led""Itowe-'ef,lCIu:al e....de~ from .nll,,:al upenme"u for llle U1>1eDCl: of Ihrnhold dOlo(1t,,,ls for lumour promoter'J .. scam: and ROI ddirut"" (V.o Duurm tr 01..1973h; Bums .nd Albe:n. peM<>O.llI communicalion). We bel;""'e tb.t lhlS is Icruci.11 issue Ind merils mucb funber study befo... ""e can at<:epl thIS as apnllclple III q~nlll.li"e IIsk asses,mellf.
Althoua)l 11 11 111.le lhal lumour prOmOler1. III C<llllr.lSl 10 illlllator1...quI""'l"'atod.nd prolonJl'd .pplicalion louen lhe,r e!TKtJ. thi' does ROllleun.rilyproVllk a ..>dema'lIlI Oharel)". MlJly SUbsllflCO$lhal '.-e .",wncc:rnod "'"h ",,11he uK<! by humal1S for prolonJl'd period, of 11.... (for example. food aOllll"'esand pesticides), and mao) of lhem .... ellhtr ROI boodo&radablo .nd ..ill.lbeml>"'. P<'nISI 'll Ille "'''lrOnmmt, or lboy mI) be orored In tllSUCS forptolOloJl'd pmocb of IJll>O (for eu.mplo. PCBs and Isbn,,),,).
Wnh~ 10 lb< poteDC)' of lWllOllf promotcn '" cancer IDducuoG. 11 IS
1DIp<>rUD1 10 ....pbascx fhal DWl)~ &I1llI>I1 lumour ~cn ....Ulmt'ld) porml. for uamplo. pho<bol esrm aDd related aplS mUlmlUypromo1e OMBA.....lJaled~ i.kID. ..bm R'pQtedl) .ppbod .. a dow of3-4 "--"'lOb pot" appb;:auoa. BouI.."Q (1977) _pb.wed WI ·OMS.... +TPA·appbcauon IS .tloul 10 umes mott POlml lhalI'O\! BA _ OM BA' pallllllll'"
ltnnSoflumolll )WIdSIIl ~skin Wlu"alllUlldow ofOMBA plusTP ofonly 380 nmoIn mulled In 75·. incidc-na: ofpapllomu .1 18 ..uk.. 10 ac "• similar lumour yield al 18..-ceb b) l..·ja ..-eo:kly IPJl/icauons ofOM SA. alOll1of76OO nmolos of OM B.... "e... required In ..JOIliOtl. 1110 phorbol esler'J lUmo~'promotor'J.nd toloocld,n .re etree",'e at extremely low concenlratlons In nellc~lluf<'. I.e. \0-' 10 \0-" .. (Wei ostein rrDl,. 1979.: Ume..... ~t QI., 1981). Thocompound 2.3,7,8·telracblorobeo:ro-p-dioxln (TeOO) promoles ral ho-palOo... r·cinoatneslS initIated by dimethylnilrosamine when uK<! at ntremely low doses,i.e., 0.14-1 .4 1'1 kllWicc weekly fo' 7 mO"lhs (PIIOI ~t Dl,. 1980), As discusl'Odearlier ,n thIS paper (.....1", Wein'telll. 1981), lhe", ''-' ~"cral ,ndl(:lt,ons lhalrvuin h.ormortos. enhtr en<logrnou, or eJ.OlCROUI. cao act as tUffI(Mlr promoleB. H01'1J>Of>Q'bo ICII1 ut.-.mdy low dotes, i.e in tbe ~'" of 10 '10
10-.0 ... TltUl nena,1I lumou, pt""""e", .'" ulmt'k'ly ""totll .nd Ll ..wid be:"rona: aD rW: ISfCwllnns 10 __ lhal bcallSC I 5lIbs11na IS I IUmourproDIOlOr. luaher d-. CUI be lolorated
174 Muhods for Es'imafl/l{/ RiJk of C~mjraJ Injury
TumQlI/ promQICf'j may lIisplay lIQSC-sctIeo:Iult dfCU$ that art lIiffmm fromthose of In,batOrl O. compkt~ <amnol~ns. for ~xampl~. DMBA-;nili.uedmouse will not d~"~lop skin lumours ifTPA,sapplledonlyoottatmonthlyint~l'\Ials. ,,1Ieru.. apphClltions al wttkly in..,......l••n: ~ffecti~ (Bou'wtll. 1%4).~v~n ullIe mi« r=i,·~. in bolh eases.. IIIe ..me latal dose afTPA In other ..'ord..ttl( ~ff""ts of r.peated doses of tumour promot'rl do not aPlXar to be "mplycumulati,'~ and in thi...n... helo'" th.~.hald ~xposu...ituation. may ws!.Hown-eT. "I!tt 'o'al dose ofTPA,.th(f,O.n\(,n ,wo (XlXn",.nlS. w~ hav~.
'dose-5<hedul~ th...hold' rather than con,..ntional ·d<Jse..th...hold'. This aspectoftumou. promOllon <ouldcompl.,.,. quanhtati,.. nsk assessmen, m hU1lUln.,A' di5<u,sed .ba,·~. ~p,demioloai<.1 studies indicat~ that the =sat;on of~xposu.. to ""na,n (Ovlfonmental faclOrs an Iud to dttruSoed cancer mk In arelati,..ty 'hon period of lime (section 4). ,ugg~,'inlthat the action of <enainlat~ "a~ corcinog~n. humour promo'(rs) in hU1lUln, may be ....... rslbk. WhIlethIS factor also eomplicat~,'h~ ta,k of ri'k ......ment. thi. i•• comp.arati~ly
opllmillle find,ng ..l1h respect 10 cancer pm'ffit;on_ Slnet ,t .ppears that~hmination of tumour promot~rs from an ~n';ronrmnl could reduce the can",,,ri.k in a r~lati,..ly shon penod of,,1O<'. (wn ,n ,ndi"duall pr("lou<ly (xposed tothe a~n1S ,n que>tion,
ACKXOWLEDGEME1\T
Th~ authors ... indebted to Dr L. Tomati. for hIS critical r~adinl of lh~
manuscript. W( also 'hank Mrs C. fllChez for typing the manu5<ript.
7 REFERENCES
Akuati. K (l97l), Compara"'.. h",oIopcal "ud... on tho bt~ot C1l'Clnonu. of til<prostate under d,rr.reot ..."""'ment "..cumstances. 10 Doll. R.. and Vodoplj.a. 1.(Ed.,) H,,., b1,'i_nlall.,,,o><lioIu '" 1M Eli<>Jojy oj C_" '" .11"". pp. 89-\17.1"'K S<><ntific l'uhlicotion No, 7, lol<"'a"ooal~ ror Reoea"'h on Can«r.1.)'00 .
!l<.<nblum. 1. (19j.l). " 'peculative ""'I."" ,II< probable n.,ure ofpromo"nl actlOO.ndI", >i~I6c>na: In ,II< undc:"'.ndlnl of til< mechanIsm, of <-omocae...... c_... ~J __
14,471---414lle,...b1"m. 1. (1915). S<qU<tl'ial"p<CUofob<m>:al"'ICI~" .~io. In lleder. F, F,
(Ed.) C""",r' of C""'"r<ht...;o;. n~",Ju, Vol, I. Pr· l2J-W l'Ionum P..... N....·Yor~Bl"mll<'&. r. M (980). tn v'''o "00'''' 00 til< """'" of,W''''' of til< pho.hol "'''''
pot..., turno. prornot~". CRC Cril. /In' T.x.. 8, 153_234.Bl"rn\><,&. r. M.. 0..:10<. Ie B.. Dumphy, W G.. ar.! Jot.... S. (1982), Specific bindin. of
pbortlol "'I<r t"OIOf p.omo"" '0 moIlS< u,,,'" .nd tultuf<ld ""n•. In Hecker. E..F"sen,&. N. E.. KUIll. W.. M••~•. F.. .od T1uelma.o. H. 'N. (Ed•.) C"""r~iJtognt<JU
.-J BioIoIIiaJ/ £JJ«l$ oj T.,." """""'m. Pr. St9_5lS. R..-.n r ..... N....- Ynrt,Bou'....n. R. K. (196ot). Scm< bioloJioal ••pecu of .kio oamncae""'i. PrD/l' up T_~J.. 4. 207-250
C~lIular and Molecula' J/uhanisms of Tumou, Pro_lion 175
8out..·.U. R. K. (1917). Tll< rol. of iod<l<lion of OlMh,n. decarbo,~'I... in tumo,promOtlOn. In Hi.tt, H, H" Wa,.on, J D.. and W,n".n. J. A. (Ed•.) D'ilI"" ofH.""",C"""". &xli< 8. Mulu,"isms of Catei""ll"."i', pp. 17)-783 Cold Sprinl Ha'bo'Laboratory. Cold Sprinl Harbor. N..... Yo,k.
8out....ll, R. K .. Verna. A. K., Ailiendel. C. L" and A'trup, E. (1982). Mou.. ,kin: au«ful mod.I,~·"emfor studyiol ,I>< mech.n"m ofehemlCalearrino~en«i"In Heck.r,£.. FU«OII. N E.• Kunz, W., Marko, F., and Th ..lmonn. H. W. (Ed.,) Coca,d"""."",;.IJ1Ui &oIf)<Jltal Cffu" of T"""" 1'",_,,,,, Pl'. l_U Royen Pt.... N.,,", York
OH:"lI. S J" Sola. M" LL. M. H.. and ChoulO.lmkov.l. (1982), E>ophageal e......' '"Lin".n Coun'y. China: a po<>ibl••'iolOiY and mec~.n,,,,, (,n,tLo"on and p,omo,ion)In Hecker, E" FUl<oig. N E.. Kuru. W.. Mark,. F.. and Thi<lm.nn, H W, (Ed•.)C<Xc,ei""ll.""'i' IJ1Ui 8iolofi<:cl Cff.w ofT"""" 1'",,,,,,,,,,. PrJ· 1;,7 _lJ4. Ra..n P......:-Ie... York.
Colburn. N H.• former. 8. F.. N.I",n. K. A" and YU'pa. S. H. (1979). Tumor p,oRl<>«rind..", a""borage ",depend.nce "r<,..""bly. NaMe, 281, 189-591
D.lldori. G., L,nl<n. C A" B.'n ....... F. L and Ma"~·n. R. (1964) An .p;d.m,olol'"'Pl""""~ '0 the I~·mphom.. of African <hildrrn and B"rki,,', <arcomo of II>< ja.....,F<"pw. Bioi. Md, 7.431----449
Day. N. E. (1982), Ep;d.m,ologieal ",'ide""" of prOmolL"lI .tT",,,: the ...mpl. of br<'"<a""". 10 Heck.r. E" fu",n'l, N £" Kuru. w.. Marko. r.. and Th"lm.nn. H W(Ed•.) C(K=i""lIt""sis cod Bi()iagiec! Cff..u of Tumo' Pro"""",. pp,183-199RaV<n P...... N.w York
d. Se,...., F, L .nd A'hby, J, (1981), Cud""uon 0/ Siler,· Te,m Tnrs fo' C.,,!rIogt"""'.£11<"ier Non~·Holiand, N.... Yo,k: 827 pa~.
D,amond. L O·B",n. T. G.. and Bai'd, W. M. (1980), Tumor p,omo«rs and lhemechaniun of 'umar I"0mo"on. .'1.1<'. Co."" lie>" 31, 1_7.
Doll, R (1978). An .pidom,ololi<ol pe"pe<t,,,. "f'he bLololY of ..."""r. C"""', ElL... 38.lS7J~)S83.
O"edger. P. E.. and Blumberg. P. M. (1979). Quan'i..ti'·e oo'relation bet.....o io "'''0and In \1\'0 a«ivi'i« of pho,bole"er•. Cante, ElL,., 39. 714-719
Driedger. r E.. and Blumberl- P. M (l9S0). Spe<ifie blnd,n, of phorbel «tet tumo,pro"""",. 1"0<. """•. .'leU. Sfl. (is''., 77, S<>7-1JI
Enomoto. T.. So..ko, Y.. Sh,b>. Y.. Kanno. Y., and Y.masaki. H. (1981). Tumorpromote" COU'" a ,"pld and re,'o"ible iollibi"oo ofll>< form."M and mointon.""" of.Iec'''''ol <ell ",,"phnl ,n <"I,u«. P'O<. ..,•. .'let><!. Sfl. (is,,., 78. S<>28-S<>32.
Fuber. E.. and Comc:ron, R. (1980). Th. "'luentialanal~·.i.ofconce, de,..lopment AdoCo....., Rn, 31, 12S-226,
Fi'hbem, l. (1971), P''''.'lal 1"J"s"Ia! Ca,e!rlogt"S IJ1Ui Mu'ot1~"S. EPA 560 S·77001NoMnal TechnICal Info'ma"on $ome<. Spnngfield. Virlln",: ) I;' I'"~es.
F"her. P. 8.. and We,"".in. I. 8. (1980). Chemi<:o.l·vi,"1 inte'a<tion. and mul'i".paspe<" ofeoll "anuonn.,ion, In Mont...no. R., B."'oh. H" and Tom.,i•• L, (Ed.,)MoIu.!", and Ce/lldo, As~m of C<uoirto(je" Sc,,,.!Jtg Tem. IARC ScotntLlkPubl",a"on No. 2J, W. 113-1) I In"rna'ional Aleocy fo' R""areh on Ca~. Lyon.
Fujiki. H. Mon. M. and S"g;muro, T. (1981), Indole alk.lold, dib~d,o..leoc,din B.teleocld,n and I,·mlby",o.in A .. membe" of a rot,,"' elo" of tumo' promo"". I'roe."",•. AeU. Sfl. U.S.A .. 78. 3812-38J6,
F"iLko, H .. Supnuma. M., Nokoy..u. M., Hash,no, H.. Moo.... R. E.. and SUILmU,". T(1982). The third <la" of n.... 'uma, promoters. pol~'a«tat« (d.bromo.ph}'Iia'mmand oplysiatmin). con dilferen"ate bioloi"al ""on, ,eI,,·.n' '0 'umOl promOl<".G""". 73, 4~J""'99
Green<b>"m. E.. and W.in"em. I. B. (1981). Rele....n« of 'll< """""p' of 'Umor
176 M~11uNb fo, EJtimt;llin{j RW< of Ch~mi~tll Injury
promo"on 10 ,tl<; CllUj.ltion ofhuman "'"00<1. In fmoglio, C, M" iln~ Wolf, 101, (fW.)Prog,ess in SW"~u-al fati'alog,. pp. 21--43. MlWOn P>Jbliohin•. U.S.A.. Inc.. N.",York,
Haonszcl. W •and Kurino",. M (1968). Stlldie< of lapan... m,.",n". !. Mortal"y f,omcor><e, and othor d,scasc..mon. Jopon<sc in tn. United S,.t... J. ,."n. C"""", I"",. 40,434,
H.d". E.. fu..nll, N. E" Kunr. W. Mark•. F .. and Th,olm.nn. H. W. (1982),c.xa"~JIJ I>Jld llil)/ogicaJ £ff«" oj Tomw' Promote". R.'-.:n Prcu. N.... York:664 pal"-
Ho,delbe""r, C" and Mond.l, S, (1982). Effoct, of tumor promo.." on lbe: diff.ren_rl.tio" ofClH lOT 12 mou...mbryo fib'obla.... In Heck.r, E" Fu..nll- N. E.. Kunz,W.. Marks. r.. ,nd Th,.lmonn. H. W. {Ed.,) C""a'cU>ogeMW I>Jld lliologjoaJ EO'«.. ojT"""" f,omot"', pp. 391-3"'. R,,-.:n Pre". N.... York
Hi"'....'., T. K.hna... 1., Fujik" H. and Sulllno .... T. (1~82). A n<..' tumor·promo"nl 0l.nt, dihydro'olc:ocidin B. markedly <nh'n«> ch.n"cally indu«d malil_n.n' cell ' ...n,formation. So"nt:<, 216, l27-52~
Hi...y.ma. 1.. ood Ito. Y. (1981). A ,...,' V1<..' of the .,,0101Y of na",pbarynl••1c.reinoma. Pm', .lied" IG. 614~22.
Holl.t.,n, 101 .. McC.nn, J, Al\I<losan,o, F. A., and l"ichola., W, W. (1979). Short.."m1.... for care,nOlcn. and mO"lon•. Mulat. Rtf.. 69,133-226,
Horowill. A. 0 .. Fuji... H.. Wetn".,n. L B.. J.ff...y. A.. Okln. E.. M""re, R. E" oodSollimuTO, 1. (1982&). Compo""i,·••ffect> of .pl)·.I.,,,,in, d.bromo.plysiato"nInd ..Ieoc,din on r«<ptor bindinl Iud pho.pnolipid mc:lOboli'm, Cana, IIL, __ 43_1129_1531
Horowi1z, A. D., Gr«ll<boom. E__ Nicoloid<,. M.. Wood....,,!. K...M W.m...,n.]. B,(l982b). lnhibnion of pho'bol "'er-r«<p,o, bindinl by • foc'or from oum.n ..rumMol«, n/illial. :. 541-553,
Horo..."z. A. 0 .. G,een.baom. E.. .nd W.,n"..n. ]. B. (1981). lde""fica"on of re«p1on;for pliorbol ..tor 1umor promoters in intoc' m.mmolion ",II••nd of on ,nh'b,to' ofrecep,or bindinl in ""'I0lt< ftuid•. f,oc, /UTln. Acad. Sd. U.S.A .. 78, 2315-2319.
IAIl.C (1980). u"fj·,mn and Short-tm" S""""''lfI AS"'J",j", CaICU>ogetr.S: A C,itioaJAp/"oisal. IARC MonOl"ph. Supplem.n, 2. 1""m."on.1 AlOney for Rose.reh onCaneor. Lyon: 426 pq<>.
IAIl.C (982). CMm,cab, Indusl'iaI Proc~"", I>Jld Industr", Auo<wt<d ~'j,!I Cant:<r ...HUITt4IU. IAIl.C Monolraph" Volo"," I '0 29. Soppl.mc:nt 4. In'.mationol A••""yfor Il..... rch on Cancer. Lyon' 292 pal".
lick, H" Watkin" Il.. N., Oloon. D. l.. Madsen. S.. Ro'hman .. K. l.. Ind W.lker, A, 101,(1979). Ropl.cemc:n1 ""ol"n, ond ondmll<tri.1 CIOC<r. Ne~' Eng!. J. Med.. 300,218-222.
Kuu..klk T.. Honm•. Y...nd Ii""om,. 101 (1981). The ,umor promo,or 12.Q.' .....dec.noyl phorbol_13-=to... ,nh,bn, or ."bon«> 'M"""on of dlff....nti.'ion ofmOoS< myeloid I:.komi. cells d<p<ndi". on 'ho 1J1l< of S<T\lm in ,he medoum. GatlIt. 72,310-314.
K<ller. R. (1979). Suppr...lon ofoo,.",1 zn"1umordef.".. m«h.""m llrphorbol "'.ro,Na,",~, :82, 729-730,
1'",..11•• A. R. (1982). Elim,n.,;on of melabolic .""per."on and 'ho Ind",,'ion of oUt.r<hroma'id ..oo.n not pr<>pen... <o'nn'oo '0 In promo'inl 0' co-c.Tt1n<>l<OIc•••nt>. CIlI'C"""'< 'is, J,4'1\1_103.
Kn.donn. A. G. Jr. (1~71), MOIO,ion.nd cal1C<r: .1Z,,,,.,..1 "udyof ..."nobIWom•. Proc",,'n, Anu/, Set. u.s A ,68, 82ll--323,
KOp<bon, S. 101 .. U<hida, I.. B...nfman, A. R.. Dailey, Il. G ••nd Fei, B. Y (1976).
C~lIular and Mol~nllar Jf~chanjjms of Tumour Promo/fon 177
An"leuk.mIC pn"",ple' '001.,«1 f'om 'ophorl>w:.ae pl.n". S<~'"'~, 191. 571-512Lame. c.. G<ntil. A...nd C!Iou,ouJinkov, 1. (1974) Two''''F moli~....ntlf.n.form"ioo
of ",. ~brobl.m in ti"u< ""lto,•. No,",~, 247. 490---'l91,Lee. L S" .od W,in't.,n. L B. (1978). Tumor promo"n, phorbol ..t.... in~ibilbmdin,of
.pld<rmol ~row'h focto' to <:ellul" receptor<. Sr;"n". 202, J 13 ·31lLi. "I .. l', 1'...od Li. B. (1980). R«,nt p,oV"" in mearch on ewp""-ge.l can«r ,n
Chin., Adr-. C"""r R<3" 33. 12J-24~.
Linsel!, C A" .nd Peers, F, G. (1977), Field studies on Ii'.., cell C.rlC<r. In Hi.,.. H. H..Wot""n, J. D, and Win".n. J. A. (Ed,,) Til< Or~ins oj Human Can"r, Book A.In<iikn,. oj Cana, rn Human,. pp, 54~-ll6, Cold Sprina Harbor Lobo"tory. ColdSjlr1na Harbo', N.... yo,k,
lee.'.n".,n. W. R. (1979). Jo"""o....1 'otel'«lIul" rommunIC."on and the ron"ol of"owth, 8roch<m, biopJlJ'. Ae'a. 560, I~S,
leewen,,';n. W. R. (19&1). luoctional tnterc,Uul.. <ornrn"o"",l,oo' ,~. «n.[""",11""""brane channel. PIr)-',;oI. &e.• 61, 829-91 J
Lo, S. H ,Cornu" A M" To"""i., L" .nd B."ooh, H. (19&1). M"ta"niorily of ••"'<1> ofp,okled ........1.. <olle<:1ed ,n l,nn,lon County, , hip iooiderlC< are. for ""ph",,"1<oncer ,n Northern Chrn•. J. IUl,n. Canen /I", .. M. 33-36,
Mark', F., B<nooh. S., Grimm. W...nd Ikhwetl,d. (I ~78). Hype'pl.stIC I"n'fo'motionand torno, p,omotion in mouse epiderm's: po.sibi< ronsequ,nce, of d,,,o'ba,,,,,,, ofendogenoo. m""h.n"m, eontrollina proliferation ,nd diff'''nta''on. In SI.p. T J..Si"k. A...nd 800,.-.11. R. K. (Ed,,) Core"""""""". Vol. 2, pp, 91-116, RaY"'" Pr....N.... york,
Mark" 1'. A" .nd R,fkin<!, R. A. (1978). Erythrei<okernIC diff<r.n".llo•. A R...·Di<>c"", .. 47, 419-448.
M."onop. E. (1979). Heredrtof}' retioobla"""",; _ f""ton.nd.J' at onset. J. "'In.
Canr<r 1"" .. 63, 9Jl_'lJ9Mond.!. S.. and H.,d'llte,,.,. C (1976). Tra",fOf1OO"on of CJH-l0 I 2 CIS mo..e
embryo fibreblam by ulrra.,olet "radi.,i"" and , phorbol """. No[_, UO, 710711.
Morton. J. f. (l%8). Plan" ••"",ioted "ith .sophageal Caneer ease> i. Curacao. Can"",iIL'__ 28, 1268-l271
Murt.y. A. W.....d Fot.gerold, D, J. (1979). Tumor p'omo'.", mh'bIt """.bollocooperation in cocolto" of .p,d"mal.nd 3D edl•. Di<>clle",. bwpIrJ$. R... C""""""..'II, J9~-4(I1
Ouaz.on•. R.. Moun"" N .. Brun. J.. D",,·o., C .•nd Yomasak,_ H. (1981), EfT"'t ofptlOrbol "«r tumor promot.... on C, t~, loola,;on ,.d eha'a<t<riu."o. of TPA""i,tant rnotant " ...ins, Cold Spriltf Harbor Sympo'i"", "" C. ,I,go... , p. 71. ColdSprin, Harbor Lahorator;', Cold Sprin~ Harbor. N.", YOf~
!'e"ino, C .. Fry, R. J. M.. Staffeld, E.. .nd Chri"oph". J. p. (1977). Enh.nci.i ,ff.." ofpltenohar."on. and but;'loted h;<lro')'to1uene on 2-a«tylaminoft""",n'.'ndU«dhepatIC lomongen..i' in the ..." Fd C""",'. To,,, l~, 93-%
p.to, R, (1977). Epid,m'olol)-', mul'i"'!!e mod.I, and short·«rm rnu'"ll<nicrty t..... InH,o". H.H , W..""n. J. D...nd W,n".n, J, A. (Ed.,) Orif"'-' a/Human C_e!, &x>Ir C.H"""", RWc A.....""""", pp, 1403-1428. Cold Spnna Harbor Lobo,,'of}'. Cold Sjlr1nlHarbor, N.... yo,k
1',,,,,. H. C" Cold,worthy. T .. e.mpbell, H A.. ,nd Pol.nd. A. (1980), Quarttilati,.evaluation of the promo"on by 2,3.7,8.l<t""hl",ohe"",·p-d'0"n of h.patoeamrrt>1''''''' from dnne'hylru.."",m'ne. C....." ilL,,, 40, 3616-3620
Poiley, J. A., R"""n, R" ....d PI.nla, R, J, (1979). Two·.,ago mali",ont Ir.n,forma"on inham.«, embryo «n., S,. J. Canr<r. 39. 8_la.
178 M~lhodJ for EJllmal;ng RiJ* of Ch~m;col Injury
RaLOk, A,!", (1973), UI\ral\r\lCt~r~~ hl"QIQpcal ilnd b'QCbc;micAl ill'''iltiQnJ p'Q<!~hy 12-0-1'lrad«~noli pnorbol·13·;>«to" on mou", ,pllkrrni' '00 tb.u "l.""nee 10.km lumo. pmmotion C"""tf 11..... 33, ,69 278
hock. A. N .• and BUrdIy', K (191), Ull-aw"c'ur,1 ,nd b,oco.mlCal chan~e< lll<l..ccd inmo"", opid.rmi. by , n,JlO'pl,,,", 'g,ol. ethylphen)ipropiolot•. C"""" Res.. 33,2221-2,)(1.
Reddy. B, S.. \\I."bu,I". J. H., all<l Wynder. E. L. (1978), O>lon ",nee. bile ..It, a.'''mor promolers. In Slop.. T J., SI"~k, A...nd 8ouI".II. R. K. (Ed•.) Ca,d,.,;og..""".VcJ.2. M....Iwn""'" D/ T.....," PTa""'ic/I 0JUi Cocauini>g..,...'i>, pp 4~3---464 Ra",nP'elI!, No,,' York.
Sell, M .. K""o,,,. M .• 'nd Mo"uy·o",. T (l%9), C"""" Ma.ldlil! for s<1"cI<"d SOl... in24 Coo",.;'" No.j 1/'J64-/9(,J!. Send... Dopanm<nl of Public H,al'b. TobokuUni""I,y $obool of Med""n•. Tahoku
SehkolI, I. J.• and Hamomnd. E. C (1979). """"0> '00 .moki.I{Ed,lorral). J. A"'.-.I.A .... , 141, 4~8-4j9
Shlnozuka. H.. Sell•. lo-t. A.. Kllyal, S. l.. Sell. S...nd Lomba.d,. B. (1919), EII«;" of achoh,.. devoid dlOl on ,bc; ,mergonco of ,lpha·Vu',myl.,ranspep'ida.e-posi"". f"""n,ho Ii"r of carcinoi.n.tr• .,ed -a". Co1IIC<". 11...., 3\1, 2S15-2~21
Sbo)·,b. M.• and Tooaro, G. J. (19W). Speclfi< bip. affinity cell mombran. r=p'o" forbioloiicaUy at,,,,. phn,bol and ,"g.nal "'<ro. Sa,.,.. , lA, 4~l--4~~
Si"~k,A., and Tu, A. S. (19W). Cell <ulture '"mOr pmmOl1On "pe""",n" "Llh saccharin.phorbol m)',,"". ,ee'ot. and ""'eral Cammon food .,.«n,I•. Co1IIC<"' L<"II.. 10, 27-32.
Slap.. 1. J.. S,,',k, A , and Bo"lu'.n, R. K. (1978). Caui>ll>g<....i>, VcJ. 2. M'cIwn""" ofTw>w' Pro..."ic/I 0JUi Cocau..<>g......i>. R..·... Pre.., N'''' '{ o,k, ~~8 pai<".
SUi,mu-a. T.. Fuj'h H., Mori, M, Naka,·"u. M T.,da. M.. Um..."·" K ,ond Moore,R. E, (l\l82). T.lwc>d,n. n.... naturolly occumnl 'Umor promo"r. In Heck". E..F.......ii. N. E.. Ku"-,. Wn M"ks. F" ond Tnl.lmann, H. W. (Ed•.) Co<a.cini>gtr><".u0JUi 8~u:al Cff..w of T.""" Pro",,,,,,,, Pr. 69-13. Rav.n Pr.,•. N.,,· Y",k.
Tm.ko. J E.. D.w,,"n. B.• Yolti. L, P, and Ch.ng. C. C. (1980). Saccharin may aCla..'umo, promo"r by' inh''''"n, ",...bollC cooperation bet.....n ""II., Sa"", U1!.109-110
Trasko, J. E.. Yoltl. L P.• Warren, S, T.. T.u.hLmo'o. G...nd Chang. C C (191)2).Inb,bI,ion of ",,11---«11 commun",ation 1» '"mo. p,omore". In Hech,. E.. F"""'il.N E.. Kunz, W., Matk... F.. and Tn..lmann. H W. (Ed•.) COCO"""'fl""#J oJId8icJ<>gical Cffw, of T."",r P'''''''''m, Pr. 565-5SS. R.,·... Pre... !"'" York
T.'o. P.O. P. (191)1) n.. "udy of'.... rnocbaniun of i" ,i"o noopla;tic Ifan.forma"on ..related '0 cane., ,,,k ..........nl. In Ricbmond, C. II .. W.I>h. P. J.. .nd Copenha,·cr.E. D. (Ed..) H..a1,~ Ri>k Analy,"" Pr, 313-3,6. Fraokhn 1",i,." Pre... Phllad.lphia.
Tu)·n•. A. J (1979) Ep,d.mlalalY of ,I<onal .nd ""neer Ca"a. 11.".. 39. 2&40-2&44Umed•• M., Noda. K.. and Ono. 1. (19801. Inbibi'i,," of ....'.bol1c cool><ra"an In
Cbi.... h.m"" "II. by ,...';ou. oomjlOulld, includ,nl IUmar promo..". GOIl1I. 71.M4 ~20.
U......w.... K . W.in""n. I. B.. Harowiu. A .. Fujiki. H., M,,,u"'~'ma,T.. .n<! S"pmura.T (1981), Slm,I",,)' of "leocidin B ond p~orbol ..Ier '.mo'" promo«" in <11'«;" onmombr'!l< 'ecepta". Na..... ,:I9O, 411--41l.
Van Duu,..n. B. L. K.LI. C .•nd Goldscbm'dl, B M (197),). Cocaremoa...ic ai.n" intobacco "''''IOogene.I•. J ""'", COI'C" 1""" ~1, 703-705,
V.n Duuren, B L.. S"·ak. A .. Sopl. A .. Seidman, L and Kat:. C. (1971b) 00.. respon... "udl"wi 'ho pu re 'Umo,_promo"nl 'l.n" pho'bol my';"." acet.1< C""",.Rrs.. 33, 1166-2172-
Woo." J.. .nd H«ker. E. (1978), Cocarc"nOl.n. of tho dllC'P"'" "ler 'YPO from erolon
C~I/ljla. """ MoIu.,lllr MuluIniJnu Ilf Tumour Pr<>MOIWtr 179
fta,..n, l. and «<>pba;U1 cancer in C"ra<c&<>, Expn~n'"" .14, 679---{)8JW,.n,..,., I B. (I~SO). Elf"''' of phorbol ..",n in 0011 cullU....d I~," ,,1<>,,0<0 to <hon
torm a,..)', fortumoT promo,.n, In M"h••. 1'., Dunkel. V., and M,~lm",. M. (Ed.,).fdr"""" ;" Mrx/r.n E""i._nllu Tox ir<>Jogy. V"'. I. M""""",,,,,, C,II T'oN/"''''''' i<Jftby CItr",;.:,u C....,~, pp. 427---<1016. Seno', l'rew. 11'IC.. f'n",.""". NJ
Weill""n, I B (I 'IS I). S'ud>es on ,he m.dran"", oforuon of,,,,,,,,,, promo'm &nd ,he"........."'" 10 ....m....ry CO""""",,,..,•. In McGrath. C M.. Brmnon. M l nd Ibcb.M A. (Ed•. ) C.11 B;"Iogy_ BU,,",I Ctmrrr. pp. 42S---<lSO. Aca&mic l'rew., N Yor~
W",n.t.,n. 1. B.. loo. l. S.. F,.he,. P. Bu Muf""'. R, A., and Y..........,. H (1979.)Act"'" of pho'boI ",'m in ",11 cultu,,' m'mICry of 'ran'forma""".•l..,od d,ff,rm·tia'ion and off"''' on 0011 ""'lObran••. J. ",p",,,,,,I,,,. S'h'''' 12, 195-208
W••n".,n. I. B.. WLJI.'. M.. and Pl<tropoolo, C (1977). Tho "",ion oftumoT-prolOotin.a..,n" ,n ",Ucult.". In H,a", H. H.. W."on, J D.,.nd Win",n.J It. (Ed•.) O.ig""o/H"""", c"""'. lJooJ:. B. M"IIO'oiJ"" 0/ CO.dllll(J.....,., pp, 7S1 -172, Cold Spnn.H.rbe,- Lobo,",o')', Cold SpnrLJ Harbor, N... York.
w..n"eill. I B.. Yama..ki. H.. W~r.l>L loo. l. S., F,oh<,. P B.. Jelf,"}', A. M.. andGrnndbe,..", D. (1979b). M<>l=1ta, and «Ilulo, " ..n" O"""UI1N ..lIh tho ....ion of,n,".."n...rrinovn••nd 'umo, promoton. In Grillin. A. C., and SIa.. , C. R. (Ed.,)Corrilwgot<r-ld"IIific0l;"" oNi M"IIO'oismJo/.f<l;"". pp l'l'9---<1IS R..,·.n P...... N...Yor~
W,II,am,. G. M Telan•. S., and To••. C (1981). Inbib,"on of int.",,1Iul", Commun,·c.."on bel"''''' h"., 00110 by ,1>0 I,,·., tumo' promoto, 1.t,1·lfI<hlo,.,.2.N".(p""hIo,ophrn)'I) <than<. Ctmrr, Utt., II. ]3~-)44
Y.....moto. N., .nd zu, H.us<n, H. (1979) liI""" promo,., TPA cnlaf>«O ,,..,,,fom>·a"on of ~u....n l<ukOC}'I" by Epo,,,,n·lla,, ",,·uo. Norun 180.244_245.
Yarna..h H (1980). R"'e,,,bI< uwhou"" of <><11 dilf."nuotlOn bJ-' pho,boI ..'m .. apossible """han;"", of pr<>moIion "'I' ,n ch<m.:al .....,noron<>i<, 10 Moo'....no, RIla,,""~, H., ond Toma",. L. (Ed,.) ,\l~ oNi C.//ukl• .flp«tS 0/ Cot<~S<,,,n~Te"'.IARC Sci<n"lic Publ",a"on No. 21. pp. 91-111 Int<mationaIA..,ncrfo' R rch on C01'lC<r. Lyon.
Yama ki. H.. 0'",'00. C.. and M."oI. N (l911la). In ,.;,,,, ".dies on tho """han;"" oftumor p,omot.,·m«!,otN tnh,bLuon of «II d,ff".nl,..uon. In H",k.,. E., Fuson,..N. E.. Kuna. W.. Mark" E. &nd Th<11....nn. H W. (Ed,.) C«<mIllll(J<'''''''_BwIo9'<tJl Ed"" o[ T"""" """""'m. 1'1'. ]59-)77. R....en P..... N.... YOfk.
Y.......h H.. O",·on. C.••nod Manol, N (19112b). Sp<clk bLnod,nl of p!Io'boI ..tero toFri<nd c')'I~'oku~cmio <><lIo-l<lI"r&l propcn.... do..........Iou,," ond "IOlionolup 1o«11 d,lf...n"ot""'. Cot<lIIIl(J<'tot.is. 3. 'l()$-910,
Y.......k,. H.. Enomo,o, T.. Ma".I. 1' .. Sh,h>. Y.. ar.d Kanno. Y. (I983). Tumo'promotor·mod,a,ed ",."ibl. 'nh,blti"" of <><1I-e<1I commu"",a"on (.I«trical ,,"up'lin.: t<l..uon.bip "'nh phorbol ..t" bind;n. and <k 11<>1'0 ",",,'omol«ul< 'ynt"'i,)~p. C.II R.s.. 146, 291-308
Yamasal:.,. H.. F,ba<~. E.. N\ld.<I. U.• W",o,,,,,n, I B, R,flc,nd. R A ond Mart" P. A(1m), TUlllOf promol.n ;nhlbu 'P""........... ond 'nduc<d d,lf""n,,..,,,," of mu"ne<f}',h,ol<uk.."•• cell. ,n ~I,ur< hoc. lIOin. .f<ad. xi. L'.S..f. 1~. 3451 - 34SS
Yomau"i, H.. Sa,nt·Vi1'lC<n'. L. .nd M.ftCl, I' (1980). Lon,-'.nn olf"'l of .. 'urnnrpromotor. 12.Q.'<tradocano)·1 phor\>ol·IJ.a«..to, on ,nduc<d d,ff.,.",io,i,," ofFn.nod ku~crni.a cello. Ctmrrr III'. 441. )780 318S
Y.ma..kt. H., Worn"cin, I B, Fibach, E.. Ri1\'nd, R. A .. and Mar~,. P A. (1979).Tumo, promot.,·,nduc<d ad...ion of ,be OS- 19 0101'1< ofmu"". 'rythro!<uk.mia c<11,.C u /I", J9, 1989-1994
Y ko, H, WriM",n. I. B.. ar.d Van O"ur<n, B. L, (1981), IndtICt"'" of <f),th.
rolcuktmia "II adhnion by plam dllClpl:n~ tumor promoter1, Ca,cilwgrlll'Jjj, ~.
5J7-54J'{ on" L. P., Ch.oni. C. C" and TroskoJ E, (1 919). Et.m".atoon or mtlabohc<:oop<rot1on
.n Chi""'" ham"er <:<U. by a tumor promoto,. Sclto<" 206. 1089_1001Vu.pa. S, H., and Morpn. 0, L (l'lllI). Mou", nln ,,,,It. ,..i"ant to tenm",,1
different;alion a.sociated "i'h ;n.tio.uon of cardnov"".i•. N~,"". 293. 72-74zur Hauoen, H., So'nkamm. G. W.. SchmKl', R.. and Hocker, E. (1979). Tumor in;,iators
and promot." In the induction <>fEpo<em-Elarr vi",•. p,«. "",n. A<aJ. 5<1. U.s.A" 76,)&2_185
