Cellular and Molecular Mechanisms of Tumour (1)

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Cellular and Molecular Mechanisms of TumourPromotion

T H O MA S J SLAGA

The University of Texas System Cancer Center Science Park-Research DivisionP.0 Box 389 Smithville Texas 78957 U.S.A.

I Introduction

I Mouse skin two-stage model of carcinogenesis

III Two-stage versus com plete carcinogenesis

IV Cellular and molecular even ts in mouse skin tum our prom otion

V Inhibitors of mouse skin tumour promotion

V Suggestive mechanisms of promotion from cell culture systems

MI Multistage promo tion: a unifying m odel

VIII Conclusion

Keywords Skin carcinogenesis, initiation, promotion, phorbol esters, freeradicals, inhibitors, prostaglandins, polyamines, d ark keratinocytes, differ-entiation

Summary

Tumour promotion appears to play a critical role in carcinogenesis in anumber of t issues. Some tumour promoters such as the phorbol ester and

teleocidin type promoters appear to act through a membrane receptorprotein kinase c?) whereas other types such as benzoyl peroxide may actthrough a free radical mechanism. In both cases important membranechanges occur. T h e ma jor effect of tum our pro m oters , regardless of th e type,is th e specific expansion of the initiated cell population in a target tissue. Th is

appears to occur by direct and indirect mechanisms. The skin tumour

prom oters, possibly by mimicking som e endogen ous growth factor, app ear t ohave a direct effect on the initiated cells which leads to their decreased

differ entiatio n. A n inhibition of cell-cell comm unication an d stimulation ofdifferentiation of non-initiated cells appear to be important indirectmech anisms of furth er expanding the initiated cell population. num ber ofothe r cellular and molecular effects of tum our prom oters such as stimulationof prostaglandin and polyamine synthesis, gene amplification andchromosomal changes also appear to play a role in tumour promotion. The

studies which reveal that tumour promotion can be operationally and

Cancer Surveys Vol 2 No 983



mechanistically further divided into several stages suggest that many of the

cellular an d m olecular even ts occur in a specific seque ntial ma nne r in which

certain inhibitors ar e very specific for certain stages of p rom otion.

I Introduction

Tumour promotion research has accelerated at an explosive rate during the

past deca de an d continues to d o so because of i ts importance in th e unde r-

standing of the induct ion of human cancer . The promotion process, being

reversible for a relatively long period, perm its the investigation of effective

means of preventing the induct ion of cancer Slaga, 198 0). Th e extensive dataavailable as well as the multistage n atu re of tu m our prom otion suggest that

this process, which is now thou ght to occur in most t issues whe re cancer can

be ind uced o r where it occurs sponta neous ly, may involve the interaction of anum ber of endo geno us factors as well as environme ntal factors such as

chemicals, radiation, viruses, and diet and nutrit ion, thus unifying all the

current areas of cancer research Slaga, 1 980). In human cancer , smoking,

asbestos, radiat ion, hormones, an d diet and nutr it ion, just to mention few,

ar e now thought to have mo re of a promotional inf luence on the m ultis tagecarcinogenesis Slaga, 198 0).

As discussed in the chapter by Pitot in this issue, there is a diversity of

chemical agents that act as tumour promoters in the various systems where

initiation-promotion has been shown to occur; howe ver, much of ou r knowl-

edge ab out the cel lular an d molecular mechanisms of tum our prom otion has

com e from studies using the phorbol ester tum our prom oters in the m ouse skintwo-stage model and in various cell culture systems. For this reason. I shallconfine my discussion mainly t o these studies, particularly the skin see Slaga

t 01 19 81; Blumbe rg. 19 81 ; Weinstein , 19 81 for ful l reviews).

Mouse skin two stage model of carcinogenesis

Skin tumou rs can be induced by the sequential application of a subthreshold

dose of carcinogen initiation stage) followed by repetit ive tr ea tm ent with a

noncarcinogenic promote r promotion stage) . T h e init iat ion phase requiresonly a single application of either a direct or an indirect carcinogen and is

essentially an irreversible ste p, while th e prom otion phas e is initially revers-

ible, later becoming irreversible. A single large d ose of a carcinogen such as7,12-dimethylbenz a)anthracene D M B A ) is capa ble of inducing skin

tum ours in mice. Papillomas occur after a relatively sho rt latency period 10

to 20 weeks) ; carc inomas develop af ter a much longer per iod 20 to 60

weeks) . I f th is dos e is lowered, i t becom es necessary to administer D M B A

repeatedly to induce tumours. Even lower subthreshold) doses of D M B A

may not give r ise to tumours over the l ifespan of the mouse Boutwell, 19 64 ).

If either croton oil or a phorb ol e ste r such as 12-O-tetradecanoylphorbol-13-ac eta te TP A ) is subsequently applied repetit ively to the backs of mice pre-

viously given a single subthreshold dose of DMBA for initiation, multiple

papillomas appear after a short latency period followed by squamous cell

carcin om as after a much longer period. Th e repetit ive application of the pro-



mo ter w i thou t ini tia tion by D M B A usual ly does no t g ive ri se to tumours o r

produces on ly a few. bu t never in a dose-dependen t m anne r S laga, 1980 ) . If

mice receive a sub thresho ld dose of a carc inogen such as D M B A as in i t ia to r,

there is a very good dose response us ing T P A as the p rom oter S laga er u l .

1981 a) . L ikewise, there i s a very good do se response with be nzo a)pyreneBP) o r D M B A as tu mo u r in i ti a to r s wh en th e p ro m o te r d o se is he ld co n s tan t

Slaga et u l . 1981 h) . Th e o rd er o f t rea tments o f th e ini t ia to r and p rom oter is

a lso impor tan t . I f repet i t ive app l ica t ions o f the p romoter are admin is tered

before in i t iat ion, no tumours wil l develop.

T he main fea tu re dem onst ra ted by t he two-stage carc inogenesis system in

mo use skin is the irreversibil i ty of tu mo ur in i t iat ion. A lapse of up to on e year

between the applicat ion of th e in i t iator and the beginning of the pro mo tert rea tment p rov ides a tumou r response s imilar to that observed when the

prom oter is g iven only on e week fol lowing in i t iat ion Boutwell , 19 64 ).

Unlike the in i t iat ion phase, the promotion stage is reversible, requir ing a

cer ta in f requency of app l ica t ion in o rder to induce tumo urs Boutwel l , 1964) .

Th e tum our ini tia tion s tage appe ars to be an i r reversib le s tep that p robab ly

involves a somatic mutat ion in some aspect of epidermal d ifferentiat ion

Slaga, 1983 ) . Ex tensive data have revealed a good corre la tion between th e

carcinogenicity of many chemical carcin ogens an d their mut age nic activities

S laga, 19 80) . Most tumour-in i tia t ing agen ts e i ther ge nera te o r a re m etabol i -

cally conve rted to electrophil ic reactants , which bind covalently to cel lu lar

D N A and o ther macromolecules Mil ler and Mil ler, 1976) . P rev ious stud ies

have demonst ra ted a good corre la t ion between the sk in tumour in i t ia t ingactiv i ties of several polycyclic ar om ati c hydroc arbon s an d their abil it ies t o

b ind covalen t ly to D N A Slaga et al . 1 9 8 2 ) .

Slaga a nd Klein-Szanto unpub lished results) have recently found tha t skin

tumour in i t iat ion probably occurs in dark basal kerat inocytes, s ince a good

correlat ion exists betw een th e degre e of t um our in i tiat ion a nd the nu mb er of

dark basal kera tinocytes p resen t in t he sk in . Th e dark basal kera tinocytes a re

prese nt in the skin in large num bers during embryogenesis , in mod erate num-

bers in the new born, in low num be rs in young adults . and in very low num bers

in the old , which suggest that the se cells may be e piderm al stem cells Slagaand K lein-Szanto , 1983) . T h e ini t iat ing potential of mouse skin decre ases

with th e a ge of th e m ouse t o the poin t tha t i t is very diff icult t o in it iate mice

greate r tha n on e year of age when the nu mb er of da rk basal kerat inocytes is

extremely small .

I I I Two stage versus complete carcinogenesis

In general , as shown in Tab le 1 , mice ar e more sens it ive than ra ts and hams-

ters t o sk in carc inogenesis by e i the r th e com plete carc inogenesis p ro toco l o r

by the in i tia t ion-promot ion p ro toco l S laga, 1983 ) . T he complete car -

cinogenesis protocol in mice gives rise to a sma ll nu m be r of pa pilloma s fol-

lowed by a h igh incidence of squamous cel l carcinomas, whereas the

ini t iat ion-promotion protocol g ives r ise to a large num be r of papil lomas fol-

lowed by a h igh incidence of squ am ou s cell carcinomas. Both the com plete



l ble Cornparison of cornple te carc inogenes is and in i t ia t ion-promot ion in ~ar iouspecies"

Specre\ retrr:tzent

Mou\e Comple t ewo-\rage

R a t Comple t ewo->tage

Hamsre r Comple t eTwo- \ r age

' S e e r e ti ew h Slapa rr trl (198 la

carcinogenesis an d ini t ia t ion-promotion protocols in rats give r ise to basal cellcarc inomas an d very few papi l lomas and sq uam ous ce ll carc inomas . The

com plete carcinogene sis protocol in ham sters pro duc es mainly squ am ou s cellca rc inom as and som e m e la no ~n as he reas the in i ti a tion--p rom ot ion p ro toco lproduce $ mainly melano mas .

T he re is qui te a difference in th e sensit ivi ty to two-stage skin carcinogenesisin ca r ious s t ra ins and s tock5 of m ice such a s S E N C A R and C5 7B L/6 m ice.The SENCAR mouse i s very sens i t ive to two-s tage and comple te car -

c inogenes is . Howev er , C '57BL/6 mice ar e very ref rac tory t o two-s tage skin

carc inogenes is by B P- TP A . Even high in it ia t ing do ses of B P (1 60 0 nmoles)and high p rom oting d oses of T P A (1 0 pg) ar e ineffect ive in causing skintum o urs (S laga , 1983) bu t C5 7BL /6 m ice d o r e spond to com ple te ca r-c inogenes is by B P (Slaga , 19 83) . This uneq ual suscept ib i li ty to com ple te a ndtwo-5tage carcinogenesis within a stock o r strain of mice strongly suggests thatthe prom otional phases of com plete a nd two-stage carcinogenesis a re dissimi-lar . In a ddit io n, differences in sensit ivity to init iat ion a nd p rom otion betw eenmice ma\- be du e to al terat io ns in th e pro mo tion ph ase of two-stage carcino-

genesis . In this rega rd, 7 . J . Slaga t r a / (unpub l i shed da ta ) have obse rved tha t

benzoyl peroxide is an ef fec tive promo ter in C5 7B L/ 6 an d S E N C A R mice .The lack of abi l i ty of 7 'PA to induce a sustained hyperplasia (Davidson and

Slaga, unpublished data ) m ay explain i ts relat ive ineffect iveness a s a promo-ter in C57BLl6 mice. These resul ts suggest that benzoyl peroxide may be abroader spec t rum p romoter than T P A in var ious st ra ins and s tocks of mice as

well a s in ot he r species including ma n.

IV ellular and molecular events in mouse skin tumour promotion

Although the phorbol es te rs a re the most potent of the mouse skin tumourp r o m o t e r s , a wide variet of othe r com pou nds have bee n shown to hav e skin

tumou r-promo t ing ac tivi ty as shown in Table 2 . Following the phorb ol es te rsand dihydroteleocidin B anthra l in is the most potent tu mo ur prom oter of the

compounds l i s ted in Table 2 . Van Duuren and Goldschm id t (1978) haverepor t ed a fair ly extensive s tructure-act ivi ty s tudy with anthral in and



Table 2 . Skin tun lour p romo te rs

Croton oil

Certain phorhol esters in croton oilSome synthetic phorbol es tersCerta in euphorbia la t ices7 - B r o m o m e t h y l b e n z ( a ) a n t h r a c e n e

Teleocidin A

Anthralin

C h r y u r o b i n

Extracts of unburned tobacco

T o b a c c o s mo k e c o n d e n s a te1-Fluoro-2 4-dinitrohenrene

Be n z o (e )p y re n e

Benzovl peroxide

Lauroyl peroxideCertain fatty acids and fatty acid methyl e5ters

Certa in long-chain a lkane\

A number o f phenol ic compounds

Surface active agents (sodium lauryl culfate, Tween 60

Citru s oils

Iodoncetic acid

strong

strongqtrong

\ t rongstrong

s t rongh

mo d e ra te

mo d e ra te

mo d e ra te

mo d e ra temo d e ra temo d e ra temo d e ra te

mo d e ra teweak

weak

weak

weakweak

weak

See review by S l q a r 111 1 9 8 1 a )" Teleocidin ha\ promotin g activity :it dos es similar to T P A I. . Slapa anid 7 Sugimul.;~.

u n lx ~ b l i s h e d a t a )

der iva t ives . L ikewise , Boutwel l and Bosch (1959) have repor ted a

s t ruc ture -ac t iv i ty s tudy of a number o f phenol ic compounds tha t a re weak

pro mo ters in compar i son t o th e phorbol es te rs and an thra l in . Al thoug h

seve ra l o the r compounds i n Tab l e 1 have mode ra t e t o weak ac t i v i t y a s

tumour p romoters , no ex tens ive s t ruc ture -ac t iv i ty s tud ies have been

pe r fo rmed .

Ben zo (e )py rene and benzoy l pe rox ide have a l so been fou nd t o b e r e la ti ve ly

good t um our p rom ote r s (S l aga t a/ . 197 9 ; S laga t ul. , 1981 b) . In add i t ion ,

Sc r ibne r and Sc r ibne r (1980 ) r epo r t ed t ha t t he mode ra t e comple t ecarcinogenic activity of 7-brom om ethy lbenz(a )an th racene w a s d u e t o it ss t rong prom oting act ivi ty and w eak ini tia ting act ivi ty. Also sho wn in Tab le 2 ,f ree rad ica l -genera t ing compounds l ike benzoyl perox ide a re good sk in

tumo ur p rom ote r s . Thes e agen t s c an be cons ide red t o be ' c l eane r' p rom ote r s

than T P A in t ha t t hey have no t been found t o have sk in t um our i n it ia ti ng o r

complete carcinogenic act ivi ty (Slaga t a/ . 1 9 8 b ) .

Cur r e n t i n fo rma tion sugges t s t ha t sk in t umour p rom ote r s d o no t b ind

covalently t o D N A a n d a r e n o t m u ta g en ic b u t b ri ng a b o u t a n u m b e r o f

impo r tan t ep igene t ic changes . In add i t ion to causing in f lammat ion andep ide rma l hype rp l as ia : t he pho rbo l e s te r s and o t he r t umou r p ro mo te r s

produce severa l o ther morpholog ica l and b iochemica l changes in sk in , as

l is ted in Table 3. Of the observed phorbol es te r - re la ted e f fec t s on the sk in ,

the induc t ion of ep idermal cell p ro l i fe ra t ion , o rn i th ine decarboxylase ( O D C )

and subsequent ly po lyamines , p ros tag landins and dark basa l kera t inocy tes



T a b le 3 Summary of morphological and biochemical responses of mouse skin to phorbol ester

and other tumour promoters

Induction of inflammation and hyperplas iaIncrease in D N A , R N A and protein synthesisAn initial Increase in keratinization followed hy a decrease

Increase in phospholipid synthesislncr ease in prostaglandin synthesis

Increase in his tone synthesis an d phosphorylation

Incre ase in orn ith ine decarbo xylase activity followed by increase in polvamines

Increil \e in his t idine and DOPA decarhoxylae activityhDecrease in the isoproterenol s t imulation of C A M PDecrease in the number of dewamethasone receptorsDecrease in SO D and ca ta lase

Induction of embryonic s ta te in adult skinInduction of darh cells (primitive 5tem cells)

Induction of embryonic proteins in adult skin

Induction of m orphological changes in adult skin resembling papil lomas, carcinomas and

embryonic skin

Decrease in histidase activity

Increase i n pro tease activityDecreahe in response of G chalone in adult akin

Increase in CA MP -ind epen dent protein kinase in adul t skin resemhling tumou rs andembry onic sk in

Interaction with receptor and/or activation of protein kinase

See review hy Slaga r u l . (1981a) for individual references

C. E. Weeksrr u l .

unpublished datli

have th e best correlation with prom oting activity (O'Brien e t a l . , 19 75 ; Slagaet al .. 197 1; Raick, 19 73; Raick, 197 4; Klein-Szanto et al. , 19 80 ; Slaga et al. ,

19 81 a). In addition to the induction of dark cells, which ar e normally presen t

in large nu mbers in embry onic skin, many othe r 'embryonic' features app earin adult skin after treatment with tumour promoters (Table 3).

It is difficult to determine which of the many effects associated withphorbol ester tumour promotion are in fact essential components of thepromotion process. A good correlation appears to exist between promotion

and epidermal hyperplasia induced by phorbol esters (Slaga et al., 1974) .However, some agents that induce epidermal cell proliferation do not

promote carcinogenesis (Slaga e t a l . , 1975). Nevertheless, it should be

emphasized that all known skin tumou r p rom oters d o induce epidermalhyperplasia (Slaga, et I / . , 198 a ) . O 'Br ien e f 01. (1975) have reported anexcellent correlation between the tumour-promoting ability of variouscom pou nds (pho rbol esters as well as nonphorbol ester compo und s) and theirabil ity to induce O D C activity in mouse skin. How ever, mezerein, a di terpenesimilar to T P A , but w ith weak promoting activity, induced O D C to levels thatwere comparable to those induced by TPA (Mufson e t a l . , 1979). Raick(1973, 1974) found that phorbol ester tumour promoters induced the

appearance of 'dark basal cells ' in the epidermis, whereas ethyl-



Cellular trnd molecular nlechiit~ist~l.~f ~ ~ i t n o u rrorrloliorl 6 1

pheny lp rop iona te EPP) , a nonpromot ing ep idermal hyperp las t ic agen t .

d id no t . Woun ding induced a few dark cells, which se em ed to correla te with

i ts ac t ion as a weak prom oter Raick , 1974 ) . In add i t ion , a large num ber of

these dark cell s ar e foun d in pap i llomas an d carc inomas Raick , 1973 ;

Klein-Szanto et al . , 1980 ) . S laga et 11. 1981a) r epo r ted tha t TPA inducedabout th ree to f ive t imes the nu mb er of dark cell s as mezere in , which was th e

f irst m ajor d i f ference found betwee n these compounds .

Sk in tumour p rom oters b r ing abou t a num ber of o the r impor tan t ep igenetic

changes in th e skin such as me mb ra ne an d d i fferen tia t ion a l tera t ions a nd a n

increase in proteas e act iv ity , CA M P- ind ep en de nt protein kinase activ ity and

phosph olipid synthesis Slaga et al . , 1981 a) . In add i t ion , the sk in tu mo ur

promoters cause a decrease in ep idermal s uperox ide d ismutase and cata laseactivi ties as well as a dec rease in th e nu mb er of g lucocort icoid receptors a nd adecreased response of G , chalo ne in adult skin Solanki et al . , 1981 ;

Davidson an d S laga, 1982 ) . T he phorbo l es ter t i lmour p rom oter a nd te leocid in

A-induced changes appear to be mediated by their in teract ion with specif ic

membrane recep tors whereas many of the o ther p romoters such as benzoyl

perox ide an d an thra l in d o no t ac t through this recep tor bu t may involve a f ree

radical mechanism Solanki a nd Slaga, 19 81 ; Slaga et 11 . . 1 9 8 b ) .

V nhibitors of m ouse skin tumour promotion

A number of inhibi tors and modifiers of skin tumour promotion have been

very useful in understanding which cel lu lar and molecular events areimpor tan t in sk in tumour p romot ion . In general , the major i ty o f known

inh ib i to rs o f sk in tumour p romot ion are agen ts that inh ib i t the

promoter- induced hyperplas ia , dark basal kera tinocytes , O D C activ ity an d

polyam ine levels. and prostag landins Slaga el a l . , 1981a) . In add i t ion , sk in

tumour p romot ion can be counteracted by a number o f an t iox idan ts ,

v i tamins , and cA M P o r agen t s tha t e l eva te endogenous l evel s of c A M P S laga

et al . , 19 82 ) . Al though pu t rescine a nd pros tag land in E a n d F o n o t

prom ote sk in tumours , they effectively enh anc e T P A promot ion S laga et u l . ,

1982) . Tab le lis ts a numbe r of po ten t inh ib i tors o f mouse skin tum ourpromot ion by T P A . T he an t i - inf lammatory s tero id , tluocino lone aceton ide

FA ). is an extremely pot en t inhibi tor of phorbol e ster tum our prom otion in

mouse skin Schwarz et a l . , 1977 ) . Repea ted app l ica t ion of a s li tt le as 0 .01 pg

almost completely counteracts skin tumorigenesis . FA also effect ively

coun terac ts the induced cel lu lar proliferat ion associated with th e applicat ion

of phorbo l es ter tumour p romoters . Cer ta in re t ino ids are a lso po ten t

inh ib i tors of mouse sk in tumour p romot ion by T P A Ve rm a et al . , 1978) .

How ever , ret inoic acid was found t o be ineffect ive against benzoyl peroxide

promot ion an d complete carc inogenesis by D M B A Slaga e t u l . , unpublishedda ta ) . Verm a et a l . 1978 ) have shown that a nu mb er of re t ino ids are po ten t

inh ib i tors of T PA -indu ced ep idermal O D C act iv ity bu t no t o f

D M B A - i n d uc e d O D C ac ti vi ty . W e e k s er u l . 1979 ) foun d that a combinat ion

of F A and re t ino ids p roduces a n inh ib i tory effect on sk in p romot ion by TP A

greater than that p roduce d by each separa te ly . Th e re la tionship betw een



Table 4. Summary of inhibitors of phorbol e . ; ter skin tumour promotion"

I Ant i - in flammator) b te r o~d s ; o r t i so l , dexamethasone and t luocino lone ace ton ide

2 Vitamin A derivativeCombination of re tinoidz and anti-inflammatory agents

Pro tease inhibitors : tobyl ly\ine chloromethyl ke tone , (TL CK ); tosyl arginine methyl es ter,(T A M E ); to.;yl phenylalanine chloromethyl ketone (T PC K) ; antipain and leupeptin

5 Cyclic nucleotide.;6 Pho spho die\te ra\e inhihitors : i \ohutylmethylxanthine (IRM X)

7 Di rn e th v l \ul fo x ~ d e DM S O )

8 Rut yrate , acetic acid9 Ra c i l lu \ C ;~ l rn e t t e -Gu e r~ nBCC;)

1 0 Poly riboin o\inic: pol)rihocytidqlic acid (pol 1:C

11 Prostaglandin \ynthesis inhibitors 5 .8 .1 1 .14-eico\ii te traynoic acid (E T Y A ) and phenidone

12 l 'h ro mh oxan e \n the taae inh ih ito rs ; i rn idazo lacetophenone (R 02 2- 35 81 ) and

i rn id az ol ph en o ( R 0 2 2 - 3 5 8 2 )13 Phospholipa \e A: ~ nhi h i to r : ih rornoece tophenone

1 3 .Arachidonic acid15 Polyamine synthesis inhihitor ditluorornethylornithine, F M O )

16 ~ i \ t a m i n e ~

1 7 Dip h e n h y d ra r n ~ n e H I e ce p to r in h ib i to r ) "

1 8 Bu tyla te d I~ y d ro k y a n i \o le R H A ) a n d b u ty la te d h y d ro x y to lu en e ( B H T ) ~19 Disulfil.arn and parahydrox nni\oleh

20 Vitamin E and ch2 1 s e le n iu m"

2 2 Anti ;~ndrogen cy pro te ro n e ace ta te ( ~ p . 4 ) ~

" S e e rebiew by Slag21 l ll (19 81 3) for individual references

T J . S l a y rr 1 1 . . unpuhlibhed result\

re t inoids and cancer has been reviewed in a recent issue of Cancer Surveys

(Spo rn . 1983 ) .

The work by Belman and Tro l l (1978) a l so ind ica tes tha t p ro tease

inh ib ito rs , cyc lic nuc leo tides , d imethy lsu l fox ide (D M S O ) an d bu tyra te inhib it

mou se sk in tum our p rom ot ion by phorbol es te r s . In add i t ion to bu tyr ic ac id ,

acet ic acid inhibi ts tumour promotion (Slaga et al . 1 9 7 5 ) . T h e

phosph odies te rase inh ib i to r, i sobuty lmethy lxan th ine ( I BM X) . was a lso foundt o inh ib it tumou r p romo t ion prov iding fur ther supp or t fo r the inhib i to ry

effect of cyclic nucleotides (T. J. Slaga, unpubl ished resul ts) . Schini tsky et a l .

(1973) repor ted the inh ib i to ry e f fec t o f bac i l lus Ca lmet te -Guer in (BCG)

vacc ina t ion on sk in tumour p romot ion . I t has been shown tha t

polyr ibocyt idyl ic acid (poly I :C) has an inhibi tory effect on carcinogenesis

a n d t u m o u r p r o m o ti o n ( G e l b o i n a n d L e vy , 1 9 7 0 ) . T h i s a p p e a r s t o b e

m ed iat ed by i ts inhibi tion of prom oter- a n d carcinogen-induced cel l

p ro l i fe ra t ion . Cer ta in p ros tag landin syn thesi s inh ib ito rs , th rombo xane

synthesis inhibi tors and phosphol ipase A , inhibi tors a lso inhibi t skin tumourpro mo t ion , which sugges ts tha t a rach ido nic ac id metabol i sm may be

impo r tan t in tum our p rom ot ion (F ischer er a l . 19 80 ; and unpubl i shed resu lt s ) .Al thoug h the m echanism is no t p resen tly und ers tood , a rach idonic ac id a t h igh

doses is a po ten t inh ib i to r of tum our p rom ot ion (F ischer er r r l . 1 9 8 0 ) .

a-Dif luoromethylorni th ine (DFMO), an inh ib i to r o f po lyamine syn thes i s .



also ~nh ib it s um our prom otion which suggests that polyamines also play

a n i m p o r t a n t r o l e ( W e e k s t 01.. 1 9 8 2 ) . A lt ho ug h B H A , B H T ,

parahydroxyanisole a nd disulf iram a re poten t inhibi tors of skin tum our

promotion, their mechanism of act ion is currently not known T. J. Slaga,

unpublished results) . It is possible tha t free radicals a re impo rtant in tumo urpromot ion and thus these agen ts may preven t p romot ion by thei r f ree radical

scavenging abil i ty . Histamine and diphenhydramine inhibi t skin tumour

promotion hut their mechanism of act ion is currently not known (Fischer

et ul . unpublished results) .

V Suggestive mechanisms from cell culture systems

Extensive studies using cell cul ture systems have suggested that the primaryaction of tum our-pr omo ting ph orbol esters takes place at the cell surfa ce. T h e

expos ure of a variety of cell c ul ture systems to phorbol ester t um our

prom oters b r ings abou t an increased me mb rane f lu id ity, increased up take o f

?--deoxyglucose an d Pi an d R b+ , inhibition of EGF binding to cellular

receptors, synergist ic in teract ion with growth factors, al tered cel l adhesion

and uncoupling of / I-adrenergic receptors, an increased turnover of

membrane phosphol ip ids , and an increase in p ros tag land in syn thes is

(reviewed by Blumberg, 198 1 and Weinste in , 1981 ) . In genera l , there ex ists a

good correlat ion between the tumo ur-pro mot ing act iv ity of phorbol este rs on

mouse sk in and thei r effects on me mb ra ne s t ructure and funct ion .

Specif ic receptors with a high aff in i ty for phorbol esters have been

demonst ra ted no t on ly in mouse sk in bu t in crude membrane f rac t ions o r

intact cells from a variety of cult ure d cells an d tissues (Blum ber g er ul. 1982) .

Recen t s tud ies ind icate that . de sp i te their d i f feren t chemical s t ructures , two

new classes of turnour prom oters , teleocidin and aplysiatoxin . b ind t o the sa me

cel lu la r r ecep to r s as the pho rbo l e s t e r tumour p romote r s (Umezawa e t a l .

19 81 ; Sugimura . 1982 ) . Ho wev er , as po in ted ou t ear l ier , no t a ll c lasses o f

tumour p rom oters ac t th rough th is recep tor sys tem.

Recently , a close associat ion between the binding si tes of phorbol estersand calc ium, phosphol ip id -dependent p ro te in k inase (k inase C) has been

found. T his protein kinase is act ivated by unsatu rated diacylglycerol , which

may be t rans ien t ly fo rmed dur ing the recep tor-media ted tu rnover o f

phosphatidylinosi to l (Ashendel et al . 1983) . Castagna et al . (1982 ) have

shown that TPA can direct ly act ivate th is protein kinase in vi tro in the

absence of d iacy lg lycero l. Su hs eq ~ ~ en t l y ,iedel et al . (1983 ) and Ashende l

er 111 (19 83) have repor ted the copurif icat ion of phorbol e ster receptor s with

protein kinase C. These results suggest that protein kinase C is the phorbol

ester receptor. Because of the importance of protein kinase C in normalgrowth control, i ts activation could be a critical event in carcinogenesis in

general .

T he recen t s tud ies showing that tu mo ur p romot ing phorbo l es ters inh ib it

cel l- to-cel l communication provide an important clue to the process of

tum our prom otion, s ince cel l-to-cel l comm unication is thought t o play a



crucial role in the control of cell proliferation and differentiation

Loew ens te in , 198 1) . T he observa t ion tha t T P A and re la ted phorbol es te r

tumo ur prom oters inhibi t metabol ic cooperat ion b etwee n cells in cul ture has

recent ly provided evidence for this imp ortant event M urray and Fi tzgerald,

1979; Yottit

a l . 197 9) . Tumour-promo t ing phorbol es te rs a lso revers ib lyinhibit both the formation and maintenance of electrical coupling of cultured

hum an epi the l ia l cells En om oto t al . 1981; Yamasaki t al. 1983) . I n

addit ion to the inhibit ion of metabolic cooperation and electrical coupling

betw een cul tured cel ls , the phorbol es ter tum our p rom oters have been shown

to inhibi t the t ransfer of microinjected f luorescent probes to the neighbouring

ce l l s Enomoto t a1. 19 81 ) . A block in intercellular communicat ion m ayenhance tumour format ion in the sk in , for example , by producing adisturbance in proliferation and cell differentiation. Consistent with this

hypothesis is the f inding tha t T P A appe ars to induce the appe arance of

intercel lular spaces between epidermal cel ls in mouse skin and impairs

epiderm al cell di f ferent iation Raick , 19 73 ) .

Inhibi t ion of intercel lular communicat ion has also been shown to occurwith a var iety of known tumour promoters other than phorbol es ters . For

example , Trosko t a l . 1982) have shown that DDT, te leocidin, benzoyl

peroxide, polychlor inated biphenyls , sacchar in, bi le acids , phenobarbi tal ,

oleic acid and anthralin can all inhibit metabolic cooperation between

Chinese ham ster V 79 cel ls in vi t ro.

Many investigators using in vivo and cell culture systems have suggested

that al tered differentiation plays a cri t ical role in tumour promotion andcarcinogenesis in general . Tumour promoters have been found to induce

transiently in epidermal and other cells a set of phenotypic changes which

resemble those found in embryonic cells as well as malignant cells. Raick1974) found that tumour-promoting agents induced in basal cel ls cer tain

morphological changes resembling those found in embryonic, papi lloma a nd

carcinoma cells. Klein-Szanto t al . 1980) found tha t tumour promoters

increased the number of dark basal keratinocytes in adult skin which were

normally in high numbers in embryonic skin as well as in papil lomas and

carcinomas. Theoret ically, modulat ion of the co mm itmen t to di fferentiat ionor differentiation potential of sub popu lations of keratinocytes co uld result in

the acc um ulation of su bp opu lation s of init iated cells. Reine rs and Slaga

198 3) found tha t tumo ur p rom oters induce a subp opu lat ion of basal cells

to become commit ted to terminal di f ferent iat ion and accelerate the rate of

differentiation of co mm itted cells. Yusp a t a l . 198 2) also found that tum our

pro mo ters can indu ce subpo pulations of basal cells in cultu re to differentiate.

This could be an important mechanism in the expansion of the init iated cell

popu lat ion. In this regard, Yuspa and Morga n 198 1) found that init ia ted

epid erm al cells in culture d o not differentiate w hen e xp ose d to a physiologicaldifferentiation st imulus high calcium).The phorbol es ter tumour promoters al ter di f ferent iat ion of a number of

cultured cell types. TPA inhibits the terminal differentiation of Friend

erythroleukaemia cells Diam ond t al . 19 78 ) , chicken myoblasts Coh en

t a l . 19 77 ) , neuro blastom a cells Ishi i t al. 19 78 ) , adipose cel ls Diam ond

t al . 19 78 ) and s t imulates terminal d~ ffere nt ia t ion of hum an myeloid



Cellulur rrr~d rlolec~tl trrlecharzitrr~tof turnour prorrlotron 6 5

leukaeni ia cel ls Hu berm an and Cal laham, 1979) and melanom a ce lls

Hub e r man et I / . ,1 9 7 9 ) .Th ese effects of tum our p rom oters o n the inhibit ion

and stimulation of differentiation of a number of cell types in culture

re-enforce the importance of a l tered dif ferentia t ion in tum our prom otion . As

previously stated both an inhibition and stimulation of differentiation mayhave a role in th e expan sion of th e initiated cells in a targe t tissue.

In addit ion to the well-known epigenetic effects of tumour promoters ,

recent repor ts suggest tha t tu m our prom oters may a lso have an e f fec t on the

genetic mater ia l of cel ls . Free radicals may be the candidates for the many

genetic effects . Som e prom oters such as benzoyl peroxide spontaneously give

r ise to f re e radicals wh ereas oth ers such as phorbol es ter a nd te leocidin type

pro m ote rs may give r ise to fre e radicals by their clastogenic effect Ce ruttiet al . , 19 83 ). Oxidized lipids an d oxygen radicals are likely sub stances

induc ed by th e clastogenic effect of T P A which could have a direct effect on

the gene t ic mater ia l . Tumour promoters have been shown to cause geneamplification Va rshavsky , 19 81 ), mitotic aneup loidy in yeast Pa rry et i r l .

19 81 ), synergistic interactions with viruses in enhan cing cell transform ation

Fischer a nd W einstein, 197 9) , enha ncem ent of ir revers ible anchorage-

indep ende nt growth in mouse epiderm al cell l ines Co lburn et i r l . 1979) ,

and s ister chromatid exchange Kinsella and Rad m an , 19 78 ) .

T he epigenetic effects of the t um ou r promo ters are revers ible a nd thus m ay

be more important in the ear l ier s tages of promotion s ince promotion is

revers ib le for a reasonable per iod of t ime . On the o ther hand , the gene t ic

effects of the tumour proinoters may be responsible for the ir revers iblepor t ion or la te s tage of promotion. Table 5 summarises the various cellularand molecular events caused by phorbol es ter tumour promoters in var iouscell culture systems.

It should be pointed out that oncogenic transformation of cultured cells by

a two-stage protocol ini t ia tion-promotion) has been demo nstrated in several

f ibroblas tic cel l culture systems reviewed by Ke nned y. 19 83 ) . This is

especial ly true us ing the phorbol es ter tumour promoters in several

f ibroblas tic cell l ines such as 10 T 112 an d Balb C /3 T 3 cells . Ho we ver ,

oncogenic transformation of epidermal cells in culture by aninit ia tion-promotion p rotocol has not yet been accomplished, even thoug h

many a t tem pts have been m ade Yuspa e t ul . , 1 9 8 2 a n d T . J . Slaga et ul . ,

unpublished results) . An explanation for this is not currently kn ow n.

V II Multistage tumour promotion a unifying model

Slaga et a / . 198Oa) have recently found that skin tumour promotion can beoperationally a nd mechanist ical ly fur th er divided into a t leas t two s tages. This

was possible when they found that mezerein induced many of the cellular

events in a fash ion s imi lar to T P A , bu t m ezere in was a weak o r nonprom ot ing

agen t. Th us T P A m ust be inducing some addit ional cel lular event s) thatm ezerein could not effectively induc e. T o test this they initiated mice, gave

l imi ted t rea tm ent of TP A tha t would no t p ro m ote tumours and then t rea tedthe mice repetitively with mezerein. This regimen was an effective way to

induce turnours see Slaga et ul. , 198 0b) . Both s tages of promo tion show a good



I . ; ~ h l e S u m m a r y 01 cel lular and m olecular event5 cau5ed by phorbol ester tum our pr om otersin various cell culture \ $tern\"

Increased membrane f luidi ty

Increa3ed uptake of \a r iou\ nut r ien t \ and ion\Altered NalK A7'Pa';elnhibition of EGF hindinIn te rac t ion wirh ~ i ie r i ihrane eceptor (pro te in k inx\e c )

Synergist ic inter ;~ct lonwith growth f :~ctor\I nc r ea sed pho \pho l ip id \ n the \~~1r1cre:rhed pro \tag lan din sln hlh ~t lo n f ce ll -ce ll communica t ionAltered cell morpholopAltered cel l adhesionIncr ea\ed pinoc to\ i \Altered fuco\e-ylycopeptide\

Decrea \ed f ihronec t inI~n cou p l in g f 8 - ad rene rg i c re cepto r5De cre ase in ;icet lcholine rec ep tor

1 Srimul;i t ion . ~ n d nhibi t ion of different iat ion in epide rma l cell.; in cu l ture2 Inhil,~rion of termi nal differentiation of Friend erythroleukaemia cel ls3 Ini~i hi t ion f different iation of neurob lasto ma cel ls

lnhibition of differentiation of adipose cellsSt imulat ion of rerminal differer l tia t ion of hum an m yeloid leu kaem ia cel l\

St imulat ion of different iat ion of human melanoma cel ls

Iricre;i\ed gene amplific;ltion2 S ner:~\tic tlf fe ch will? vi ru se \3 Enhan cemen t of i r rev ers~ hle nch orag e-~ nde pen den t rowth in mou5e ep idermal ce ll l ines

Induce s i \ t e r chromat id exchange5 Induce mi to t ic aneuplo idy in >ea \ t

See review5 by Weins te in (1 98 1) and Blum berg ( 19 x1 ) for de ta i l5 concern ing thehe e f fec ts

dose-response relationship. Table 6 summarizes the important characteristics

of the first and second stages of promotion. It should be emphasized that only

one application of a first-stage promoter is required and that this stage isirreversible for 1-6 weeks Slaga p NI . 1981a). Besides TPA and

12-deoxyphorbol-13-decanoate which are known tumour promoters,

non-tumour-pronioters such as 4-0-methyl TPA, calcium ionophore A23187and hydrogen peroxide as well as wounding are effective stage I tumour

promoters Slaga l 11.. 1981a) . These agents as well as wounding increase

the number of dark basal keratinocytes stem cells?); this suggests that these

ceIls are important i n the first stage of tumour promotion. Prostaglandin E

specifically enhances stage I of promotion, whereas putrescine specificallyenhances stage 11 of promotion. Stage I of promotion can be inhibited by FA,

TPCK. and vitamin E which also counteracted the TPA-induced dark basal

keratinocytes. These dark cells normally are present in high numbers in



Table 6. Summ ary of characteris t ics of th e fir\ t and second s tages of tu niou r promotion

A good dose-response ex is t\ fo r TP A an d 12 deoxyphorbol decanoate as firs t s tagep ro mo te r s

2 The nonpromot inp agen ts. ca lc ium ionophore A2 318 7 .4- 0-m ethy l-T PA . hydrogen perox ideand wounding can act as s tage 1 p ro mo te r s

3 Only o ne application of firs t-s tage prom oter i necessaryProstaglandins are important s ince PGE: can enhance s tage I b y T P A

5 Partially irreversible

ii) Four t o six weeks can se parat e firs t and second s tages of promotion without a decrease

in tumour respon\e

b ) Th ere is an 80 1 ecrease in tumo ur response if 10 week5 \epar ate s tage 1 a n d I of

promotion6 Increa se in th e num ber of dar k basal ke ratinoc yte, (ste m cells) is im por tan t. ? 'his occurs

directly , \ t imulating ex~ sti ng ark cells to divide, converting some basal cells to dark cells , andincrea\ing the differentiation of \ome basal cell.; and differentiated cells

7 Decrea \e in lucocor t ico id recep tors hy TPA may help in proliferation of dark cells

A good d ose-r espo me e .; i<ts for rnezerein as a second s tage pro mo ter

The nonpromot ing aeen t 12-deoxyphorbol-13-2 1 6-decatrienoatean act as a s tage Ip ro mo te r

3 Multiple applica tion\ :ire requir ed

Rebers ihle for a re la ti ~e ly ong period but 1;i ter becomes irreversible5 Polyamines are im porta nt s ince putrescine can e nhan ce s tage 11 promotion by mezerein

6 Most of the biochemical event\ shown to be important in promotion occur in this stage7 Mezerein can maintain dark cell proliferation and decrease in glucocorticoid receptors but

cannot induce these events h) itself

embryonic skin (200/;,-50 of basal cells) but are present in low numbers in

adult skin < 3 of basal cells). Papillomas and carcinomas also have a large

number of these dark cells (Klein-Szanto et a l . , 1980). As previously

discussed, the dark cells appear to be the critical target cells of the tumour

initiator. and the first-stage promoters stimulate these cells to divide rapidly

by both a direct and an indirect mechanism T. J. Slaga and A. J. P. Klein-

Szanto, unpublished results).The second stage of tumour promotion is initially reversible but later

becomes irreversible. A number of weak or nonpromoting agents, such as

mezerein and 12-deoxyphorbol-13-2 4 6-decatrienoate re effective

second-stage promoters (Slaga t u l . , 1981a) . Although mezerein alonecannot increase the number of dark cells or decrease the level of

glucocorticoid receptors, i t can effectively maintain these conditions after

TPA treatment in a two-stage promotion protocol (Davidson and Slaga,

1983). Stage I of tumour promotion can be inhibited by FA, RA, DFMO,

BHA, cyproterone acetate (CPA) and vitamins E and C, which counteracteither the mezerein-induced ODC, cell proliferation, and/or as yet

unidentified events (Slag3 t t ul . . 1980b; Slaga er r r l . 1981a; T. J. Slaga,unpublished results). Based on these results with inhibitors, polyamines and

epidermal cell proliferation, as well as some unidentified event(s) appear to

be important i n stage 11 of promotion. Figure 1 depicts the various stages of



TWO STAGE PROMOTION

STAGE I STA GE I1

INITIATION IX) TPA IX) MEZ ERE INA23187 IX) DP -Tr i D

Hz02 1x1 mu l t ip le appl icat ions)4 - 0 - M ETH YL TPA I X)

WOUNDING IX)

1 DARK CELLS STEM STIMULATION OF 1 POLYAMINES

CEL LS) ARE CRITICAL DARK CEL LSTARGET PRIMITIVE SKIN

STEM CELLS)

2 PROSTAGLANDINS 2 MAINTAINED CELL

FATPCK

PROLIFERATION

HAFA

D F M OBHA

VITAMIN EVITAMIN CCPA

Fig . 1 . diagram of the various s tages of skin carcinogenesis showing the im portant eve nts in

ini t ia t ion and in Stage I a n d 11 of p romot ion and wh ere the var ious inh ib i to rs a re e f fec t ive

promo tion the imp ortant events in each s tage and where the variousinhibitors are effective.

V l l l onclusion

From this brief review one can conclude that tumour promoters cause manycellular and molecular changes in cells leading to a more embryonic

phen otype but it is very difficult to de termin e which events are critical. Th e

many epigenetic changes induced by tumour promoters appear to beimportant in the earlier stages of promotion whereas the genetic effects may

be m ore imp ortant in the late stages of p rom otion leading to its irreversibility.The alteration in differentiation induced by tumour promoters in a target

tissue ap pea rs to be th e m ajo r factor in th e expansion of the initiated cells.Although the skin tumour promotion system is a multistage process with

specific seq uen tial stages it is no t presently k now n if carcino gen esis systemsin other tissues go through a series of sequential stages similar to that in themouse skin.



C ellulur und rnolec.ular me ch ar ri~ rn .~f turnour promotior1 6 0 9

cknowledgements

Re search sponsored by NIH Grants , CA-20076 CA-34890 and CA-34962. I

gratefully ackno wled ge all my past and present technicians, stud ents , postdo c-toral fellows and collaborators who contributed to these studies.

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Cellular and Molecular Mechanisms of Tumour (1)

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