The induction of ornithine decarboxybase and S-adenosybL-methionine decarboxylase in mouse epidermis by variousclasses of tumor-promoting and nonpromoting compoundshas been studied in order to determine the specificity of thisresponse for tumor promotion. The effect of topical applications of a series of phorbob esters on these enzyme activitiescorrelated well with their promoting abilities. lodoaceticacid, anthralin, and Tween 60, all promoting compounds,also stimulated both of these enzyme activities after singleand multiple applications. The hyperplastic agents aceticacid, cantharidin, and ethyl phenyipropriolate, however,had little effect on ornithine decarboxylase activity but apronounced effect on epidermal S-adenosyl-L-methioninedecarboxybase activity. The specificity of the ornithinedecarboxylase response for tumor promotion was suggestedby the results of the above experiments as well as thestimulatory effect of a completely carcinogenic dose of7, 12-dimethylbenz[a]anthracene; a bower initiating dose hadno effect. In addition, epidermab tumors produced by atwo-stage procedure showed consistently high levels ofornithine decarboxylase activity but variable bevels ofS-adenosyb-L-methionine decarboxybase activity.
INTRODUCTION
A primary goal of research in chemical carcinogenesishas been the discovery of those cellular events followingcarcinogen treatment that are both unique and essential tothe carcinogenic process. The experimental production oftumors in mouse skin can be separated into 2 discretestages, initiation and promotion (3). This has been a usefulmodel for studying the early biochemical consequences ofinitiator and promoter treatment separately, with the aim ofelucidating the specific functional changes essential for eachstage.
The transition of many mammalian tissues from arelatively quiescent state to a rapidly proliferating population of cells is accompanied by large increases in the levelsof ornithine decarboxylase (EC 4. 1. 1. 17), S-Ado-Met2
1 This work was supported in part by Grant BC-14 from the American
Cancer Society and Grants CA-07175 and CA-05002 from the NationalCancer Institute.
2 The abbreviations used are: S-Ado-Met, S-adcnosyl-L-methionine;
decarboxylase (EC 4. 1. I .50), and their biosynthetic products, putrescine and the polyamines spermidine and spermme. These systems include rodent livers after partial hepatectomy (16, 19), the mouse salivary gland after isoproterenob administration (I 1), lymphocytes stimulated toproliferate by various mitogens (12), and some establishedcell lines in culture following various treatments (5, 10). Inaddition, rapidly growing neoplastic tissues, such as Morrishepatomas 3924A and 7777 (24), and mouse L12b0 beukemic cells (18) exhibit high ornithine decarboxybase activities and increased bevels of I or more of the pobyamines.Recently, elevated putrescine and spermidine concentrations were shown to be specifically associated with the oncogenic transformation of chick embryo cells by Roussarcoma virus (6). The mouse epidermis after promotertreatment has many of the characteristics of these stimulated-to-proliferate systems. Following a single application of an active promoter, a sequential stimulation of theincorporation of precursors into phospholipids (17), RNA,protein, and DNA (2) is observed, followed by cell division.
We have reported previously (14) the induction of ornithine decarboxylase and S-Ado-Met decarboxybase inmouse epidermis by the tumor promoters croton oil andTPA, the most active of the promoting phorbol estersisolated from croton oil (8). The increased enzyme activitieswere apparently dependent on new RNA and proteinsynthesis, suggesting that tumor promoters may functionby specifically activating certain genes (4). We have extended this work to include the effects of other phorbobesters, several promoters structurally unrelated to thephorbol esters, hyperplastic agents, and a carcinogenon the levels of these enzymes in the epidermis at earlytimes after treatment. Since promotion requires severaltreatments at frequent intervals in order to be effective,the response of these enzymes after multiple applicationsof some of the above compounds was also determined.The discovery of specific functional changes occurring inmouse epidermis after promoter treatment and a cornpanson with the effects of various classes of nonpromotingcompounds should provide some insight as to which ofthe multiple phenotypic changes caused by promotersare essential for the process of tumor promotion.
MATERIALS AND METHODS
Animals. Female Charles River CD- I mice were obtainedfrom Charles River Breeding Laboratories, Wilmington,
2426 CANCER RESEARCH VOL. 35
Induction of the Polyamine-biosynthetic Enzymes in MouseEpidermis and Their Specificity for Tumor Promotion'
T. G. O'Brien, R. C. Simsiman, and R. K. BoutweU
McArdle Laboratoryfor Cancer Research, University of Wisconsin Medical Center, Madison, Wisconsin 53706
Mass., and were used at 7 to 9 weeks of age. The dorsal hairof each mouse was shaved with surgical clippers at least 1 to2 days before use, and only those mice showing no hairregrowth were used. All animals received water and WayneBreeder Bbox (Allied Mills, Chicago, Ill.) ad !ibitumthroughout the experimental period. Mice were killedbetween 1 and 3 p.m. to avoid variations due to circadianrhythms.
Chemicals. All chemicals applied to mouse skin weredissolved in reagent grade acetone and delivered in a volumeof0.15 or 0.20 ml.
TPA was purchased from Consolidated Midland Corp.,Brewster, N. Y. Phorbob didecanoate and phorbol dibenzoate were synthesized by Dr. T. Helmes, and phorbobdiacetate was synthesized by Dr. W. M. Baird. DMBA wasobtained from Eastman Organic Chemicals, Rochester,N. Y. Tween 60 (polyoxyethylene sorbitan monostearate)and iodoacetic acid were obtained from Sigma ChemicalCo., St. Louis, Mo. Cantharidin was purchased from Nutritional Biochemicabs Co., Cleveland, Ohio. Ethyl phenylpropriolate was obtained from Aldrich Chemical Co., Milwaukee, Wis., and anthrabin was obtained from SchUchardt,Munich, West Germany. [The structure of anthralin haslong been assumed to be 1,8,9-trihydroxyanthracene. Recently, however, Segal et a!. (20) have concluded that theactual structure is the tautomeric form, l,8-dihydroxy-9-anthrone.]
DL-[l-'4C]Ornithine (58 mCi/mmole) was purchasedfrom Amersham/Searle Corp., Arlington Heights, Ill.S-Adenosyl-L-[carboxyl- ‘4C]methionine (7.7 mCi/mmole)was obtained from New England Nuclear, Boston, Mass.
Preparation of Epidermal Extracts. The epidermis wasobtained exactly as described (14). Homogenization bufferwas 50 mM sodium phosphate, pH 7.2, containing 0. 1 mMpyridoxal phosphate and 0. 1 mrvt EDTA. Groups of 4 to 5mice were treated identically and the epidermis was pooledto constitute each treatment group. Following homogenization and centrifugation at 30,000 x g for 30 mm at 0°,thesupernatants were used for estimation ofenzyme activities.
Enzyme Assays. Ornithine decarboxylase activity wasdetermined by measuring the release of “CO2from DL[b-'4C]ornithine as described (14). The substrate concentration routinely used (100 tiM) was nonsaturating, but insome experiments a saturating (2 mM) concentration wasused, and similar results were obtained. Enzyme activitywas proportional to the amount ofextract in the range 0. 1 to2.0 mg protein and to the time of incubation up to 60 mm.All values were corrected against a “boiledenzyme―blank.
S-Ado-Met decarboxylase activity was determined bymeasuring the release of ‘4CO2 from S-adenosyl-L[carboxy!-'4C]methionine as described (14). Incubationswere carried out in the presence of 2.5 m@iputrescine as anacceptor of the propylamino moiety derived from decarboxylated S-Ado-Met. Incubations without putrescine hadnegligible activity. The substrate concentration used (100zM) was nonsaturating, but in some experiments a saturat
ing concentration (2 mM) was used, with similar results. Theenzyme activity was proportional to the amount of extract added, in the range 0.05 to 0.40 mg protein and tothe time of incubation up to 60 mm. All values were
corrected against a boiled enzyme blank.The presence of inhibitors or activators of either enzyme
activity in the epidermal extracts after treatment with any ofthe compounds described below was ruled out on the basisof mixing experiments between control and treated mice,which always gave additive specific activities.
Protein Determination. The protein concentration of theepidermal extracts was measured by the method of Lowry eta!. (13), with bovine serum albumin as standard.
RESULTS
Effect of Phorbol Esters. In a previous report (14), wedescribed the induction of ornithine and S-Ado-Met decarboxylases after a single topical application of the powerfultumor promoters, croton oil and TPA. The phorbob esters,phorbol didecanoate, phorbob dibenzoate, and phorboldiacetate, have been described as moderate, weak, andnonpromoting, respectively (I). Chart IA depicts the response of ornithine decarboxylase to a single application of
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Chart I. The effect of a single topical application of 17 nmoles ofeach of the compounds tested on the activities of epidermal ornithinedecarboxylase (A) and S-Ado-Met decarboxylase (B). Groups of 4 micewere treated with either 0.2 ml acetone or 0.2 ml of one of the compoundsdissolved in acetone and were killed at the times indicated. Each pointrepresents the mean of triplicate determinations of enzyme activity(variation, < 10%). The whole experiment has been repeated twice withsimilar results. Compounds tested were: S. phorbol didecanoate; A, phorbol dibenzoate; •,phorbol diacetate. Prot., protein.
of ornithine decarboxybase and S-Ado-Met decarboxylase.The promoters chosen were Tween 60, anthrabin, andiodoacetic acid. The effect of a single application of each ofthese compounds on ornithine decarboxylase activity isshown in Chart 3A . Three points should be made. (a) Eachcompound stimulated enzyme activity at some time duringthe 1st 48 hr after treatment. A slight peak of activity(6-fold greater than control) was observed at 15 hr afterTween 60 treatment, while increased enzyme activity wasnot seen until 48 hr after anthralin treatment (1 8-foldgreater than control). After a high dose of iodoacetic acid(10 @imoles), slightly increased levels of this enzyme werefound at 3 and 15 hr posttreatment, and a large stimulationwas found at 48 hr (50-fold greater than control). (b) Thekinetics of the response was different for each compoundand each was different from the response to the activephorbol esters. In general, the enzyme activity reached apeak much earlier after the phorbol esters and fell to thecontrol bevel by 12 to IS hr after treatment. (c) The doses ofthese 3 compounds were 2 or more orders of magnitudelarger than the doses of phorbob esters used (see Chart I andRef. 14). However, these doses are approximately thoserequired to show tumor-promoting activity in a 2-stagecarcinogenesis experiment (7, 20, 21).
Chart 3B depicts the response of S-Ado-Met decarboxybase to a single application of these compounds. Again, thekinetics of the response was different for each and wasdistinct from the effect of the active phorbol esters. AfterTween 60 treatment, enzyme activity peaked at 6 hr (3-foldgreater than control) and slowly declined to the control levelby 48 hr. After anthrabin application, activity was reducedby 50% at 6 hr, but it increased to a level approximately2.5-fold greater than control at 48 hr. lodoacetic acidcaused a striking reduction in the level of S-Ado-Metdecarboxylase lasting up to 24 hr after application, yet by 48hr the activity had risen to twice the control value.
Tween 60 and anthralin had no effect on either enzymeactivity when added directly to incubation mixtures, even atconcentrations of I mg/mb and 50 @M,respectively. Iodoacetic acid, however, strongly inhibited both enzymaticactivities in vitro; therefore all epidermal extracts fromiodoacetic acid-treated animals were exhaustively dialyzed
17 nmoles of each of these compounds. Enzyme activityrapidly increased after treatment with the active promoters,reaching a peak (50-fold greater than control after phorbobdidecanoate treatment, 20-fold greater than control afterphorbol dibenzoate) at 4 to 5 hr postapplication and quicklyreturning to the control level by 12 hr, where it remained forup to 7 days, the longest time point tested. This responsewas identical in its time course to the response seen aftertreatment with 17 nmoles TPA or croton oil (14), exceptthat the magnitude of the stimulation was much greater forTPA and croton oil. The nonpromoting phorbob ester,phorbob diacetate, caused no detectable increase in enzymeactivity at any time tested. Phorbob didecanoate andphorbob dibenzoate also stimulated epidermal S-Ado-Metdecarboxylase activity, while the inactive phorbob ester,phorbob diacetate, did not (Chart 1B). Following phorbobdidecanoate treatment, this enzyme activity reached a peak(3.5-fold greater than control) at 12 hr and slowly declinedto control bevel by 7 days (data not shown). After phorbobdibenzoate application, enzyme activity was increased 2.5-fold at 5 hr and had returned to the control bevel by 48 hr.Again, no major difference in the time course of thestimulation caused by TPA or croton oil and that caused byphorbob didecanoate and phorbol dibenzoate was observed,except in the magnitude of the stimulation.
The actual amounts of these enzyme activities, measuredat the peak of activity following each phorbol ester application, correlate webb with the relative promoting abilities ofthese compounds (Chart 2). The data for TPA are takenfrom the paper of O'Brien el a!. (14). A similar correlationwas observed for various doses of TPA and the promotingabilities of the doses (14). (The response of these enzymes toTPA treatment was not affected by prior initiation. Themagnitude and time course of the enzyme activities after asingle application of 17 nmobes TPA were similar in groupsof mice treated 7 days earlier with either 0.2 @tmoleDM BAor acetone.)
Effects of Other Promoters. The effects of phorbol esterson these enzyme activities may be a unique property of thesecompounds but unrelated to their promoting action. Therefore, we have examined the effect of promoters with quitedifferent biological and chemical properties on the activities
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Chart 2. The effect of a single topical application of 17nmoles of each of the indicated compounds on the activitiesof epidermal ornithine decarboxylase and S-Ado-Met decarboxylase. Groups of 4 mice were treated with either 0.2 mlacetone or 0.2 ml of one of the compounds tested and werekilled either 5 hr (ornithine decarboxylase assay) or 24 hr(S-Ado-Met decarboxylase assay) later. Shaded bars, meanof triplicate determinations of ornithine decarboxylase activity (variation, < 10%); open bars, mean of triplicate determinations of S-Ado-Met decarboxylase activity (variation,< 10%). Promoting activities are taken from Ref. 4. Prot.,
A application of these compounds, multiple applications of
each promoter caused an early stimulation of ornithinedecarboxylase activity, reaching a peak 3 to 6 hr after thelast application. The basal bevel of this enzyme was notappreciably changed after S applications of any of thecompounds. The time course of the stimulation of enzymeactivity after multiple applications closely resembles the
@ experiment. The doses of Tween 60 and anthralin remained
kinetics of the response to a single application of thephorbol esters (compare Charts IA and 4A). Due to theexcessive wounding caused by 10 @tmolesiodoacetic acid,the dose was lowered to I smole for the multiple application
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The effect of multiple applications of S-Ado-Met decarboxylase activity, shown in Chart 4B, was not greatlydifferent than the effect of a single application. The majoreffect was to change the basal level of activity (0 hr orcontrol value). Since the doses of iodoacetic acid used in thesingle and multiple application experiments were different,it .is hard to interpret the results with this compound,
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Chart 3. The effect of a single application of iodoacetic acid, anthratin, and Tween 60 on the activities of epidermal ornithine decarboxylase(A) and S-Ado-Met decarboxylase (B). Groups of 4 mice were treatedwith either 0.2 ml acetone or 0.2 ml of one of the compounds tested dissolved in acetone and were killed at the times indicated. The epidermalextracts from iodoacetic acid-treated mice were dialyzed for 24 hr at40 against 300 volumes of homogenizing buffer; the buffer solution
was changed after 16 hr. Each point represents the mean of triplicatedeterminations of enzyme activity (variation, < 10%). The whole cxperiment has been repeated once with similar results. Compounds anddosages used were: •, 10 @moles iodoacetic acid; A, 2.2 @zmolesanthralin; @,100 mg Tween 60. Prot., protein.
for 24 hr against homogenization buffer before enzymeassays were performed. The dialysis procedure itselfdid notaffect S-Ado-Met decarboxybase activity, but it did reduceornithine decarboxybase activity to a variable degree, usually 30 to 50%. Thus, the stimulated ornithine decarboxylaseactivities after iodoacetic treatment should be regarded asminimum values.
Since promotion requires frequent and repetitive applications to elicit tumors in previously initiated mice, we testedthe effect of multiple applications of these compounds onthe polyamine-synthesizing enzymes. Separate groups ofmice were treated at 3- to 4-day intervals with I of the 3promoters and were killed at various times after the last of 6applications. The “control―groups for each promotor weretreated S times with promoter, but they received acetoneinstead of the final promoter treatment, in order todetermine the effect of multiple applications on the basallevels of the enzymes. The results of these experiments areshown in Chart 4. In contrast to the effects of a single
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Chart 4. The effect of multiple applications of iodoacetic acid, anthralin, and Tween 60 on the activities of epidermal ornithine decarboxylase(A) and S-Ado-Met decarboxylase (B). Groups of 24 mice were treated at3- to 4-day intervals with 0.2 ml of one of the compounds tested dissolvedin acetone, and groups of 4 mice were killed at the times indicated afterthe 6th application. Control (0-hr point) groups were treated as describedin the text. The epidermal extracts from iodoacetic acid-treated mice weredialyzed before assays as described in the legend to Chart 3. Each pointrepresents the mean of triplicate determinations of enzyme activity (vanation, < 10%). Compounds and dosages used were: •,1 zmole iodoaceticacid; A , 2.2 @mo1esanthralin; S. 100 mg Tween 60. Prot., protein.
although it is evident that S applications of the lower dosedid reduce the basal level of this enzyme by about 80%,which is consistent with the bong-lasting reduction inenzyme activity after a single application of the higher dose(Chart 3B).
Effect of Multiple Applications of TPA. The effect ofmultiple applications of the powerful promoter, TPA, onornithine decarboxylase activity is shown in Chart 5A . Micewere treated with 17 nmoles TPA and were killed at theindicated times after a single application or the last of 2 or 8applications.The interval between multiple applications was3 days. The control for the multiple application groups wasanalogous to the control groups in the preceding section.Little difference in ornithine decar.boxylase activity wasnoted after 1 versus 2 applications, while after 8 applicationsthe stimulation observed was much greater. Six hr after the8th treatment, enzyme activity was a remarkable 600-foldgreater than control. Although the magnitude of thestimulation was much greater, the kinetics of the responsewas virtually identical to that observed after a singleapplication. Also, no change in the basal level was seen after7 applications.
The effect of multiple applications ofTPA on S-Ado-Met
Chart 5. The effect of multiple applications of TPA on the activitiesof epidermal ornithine decarboxylase (A) and S-Ado-Met decanboxylase(B). Groupsof miceweretreatedwith I (U), 2 (A), or 8 (•)applicationsof 17 nmoles TPA dissolved in 0.2 ml acetone, and groups of 4 mice werekilled at the times indicated after the last application. Control (0-hrpoints) groups were treated as described in the text. Each point representsthe mean of triplicate determinations of enzyme activity (variation,< 10%). Prot., protein.
decarboxylase activity differed from the effect of a singleapplication (Chart SB); rather than a broad peak of activityat 9 to 24 hr after a single application, maximal activity wasnot reached until 24 (8 applications) and 48 hr or longer (2applications). Multiple applications did not increase themagnitude of the stimulation of this enzyme activity overthat caused by a single application.
Thus, we conclude that a characteristic response of mouseepidermis to frequent, repetitive applications of a promoteris an early, transient stimulation of ornithine decarboxylaseactivity. Promoters with very different chemical and biobogicab properties are all effective at doses corresponding totheir promoting doses. S-Ado-Met decarboxylase activity isalso stimulated, but a consistent kinetic pattern does notemerge.
Effect of Hyperplastic Agents. Since all promoters causeepidermal hyperplasia, the possibility exists that the increased levels of these enzymes observed soon after promoter treatment may be linked to the hyperplasia-inducingability of these compounds, unrelated to promotion perse. Increased activity of these enzymes, especially ornithinedecarboxylase, is an early response to growth stimulation inmany mammalian tissues (1 1, 16, 23). In an attempt toclarify the role of these enzymes in promotion versushyperplasia, we examined the effect of acetic acid, a
A hyperplastic, nonpromoting compound; cantharidin, a hy
perplastic, weakly promoting compound; and ethyl phenylpropriobate, another weakly promoting hyperplastic agent.In Chart 6A the effect of a single application of each ofthese compounds on ornithine decarboxylase activity isshown. The nonpromoter, acetic acid, had no effect on thisenzyme, while the 2 weak promoters stimulated enzymeactivity slightly. Ethyl phenybpropriolate caused a 6-foldincrease in activity at 6 hr after treatment, and cantharidinstimulated activity at 24 and 48 hr (3-fold greater thancontrol). Since the back of response to acetic acid was ratherunexpected, we tested the effect of multiple applications ofthis compound. However, the enzyme activity after 7applications of 10 mg acetic acid was not elevated at anytime after the last treatment (data not shown).
The effect of a single application of these 3 compounds onS-Ado-Met decarboxylase acitivity is shown in Chart 6B.The response of this enzyme was virtually identical for all 3compounds; the activity gradually increased to a peak (3- to5-fold greater than control) at 15 hr and declined to nearcontrol levels by 48 hr. The kinetics of the stimulation wasvery similar to that observed after phorbol ester treatment.
These compounds had no effect on the activity of eitherenzyme when added directly to the incubation mixture, evenat concentrations of 1 mr@t(acetic acid), 2 zg/mb (cantharidin), and 100 @M(ethyl phenylpropriolate).
Effect of DMBA. The specificity of this response fortumor promotion was further examined by determining theeffect of a single topical application of DM BA, a carcinogenthat effectively initiates in mouse skin at low doses, while asingle high dose is completely carcinogenic (22). Thus, ahigh dose presumably has a promoting component that isabsent in a bow dose. A differential response of ornithinedecarboxylase activity to high and bow doses could beindicative of this promoting component. As shown in Chart
A ofthese tumors, no consistent pattern is seen for S-Ado-Metdecarboxylase activity. In fact, one of the carcinomasexamined exhibited near-control activity, while the othertumors showed slightly elevated levels.
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Table I
Levels ofornithine decarboxylase and S-Ado-Met decarboxvlase in tumors
Tumor-bearing CD-I mice were obtained from a 2-stage tumonigenesisexperiment. The mice were removed from the experiment, left untreatedfor at least 4 weeks, and killed; their tumors were excised, and extractswere prepared. Several papillomas were pooled to obtain sufficient material. Results are expressed as the mean of triplicate determinations ofenzyme activity (variation, < 10%).
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7A , there is indeed a stimulation of ornithine decarboxylaseactivity at 2 days after a single application of 3.6 .tmoles,while no response at any time point was observed after 0.1@zmole. S-Ado-Met decarboxybase activity was also increased after the higher dose (Chart 7B); no effect was seenafter the bow dose.
Enzyme Levels in Tumors. The frequent and repetitiveapplication of promoter to previously initiated mouse skinresults in the appearance of both benign (papilbomas) andmalignant (carcinomas) tumors. If increased activities ofeither ornithine decarboxylase or S-Ado-Met decarboxylase are an essential feature of promotion, we would expectelevated bevels of these enzymes in the end products of thisprocess, i.e., papilbomas and carcinomas. The data in TableI show that this is indeed the case for ornithine decarboxylase. The tumors were obtained from a typical 2-stagetumorigenesis experiment; however, those tumor-bearinganimals used for enzyme determinations were left untreatedfor at least 4 weeks before killing, in order to eliminate anyresidual effects of promoter treatment. While elevatedornithine decarboxylase activity appears to be characteristic
Met decarboxylase activity) is associated with cellularproliferation, this is not true for mouse epidermis. Aceticacid stimulates epidermab RNA, protein, and DNA synthesis (T. J. Slaga and R. K. Boutwell, personal communication) and is an effective hyperplastic agent, but it is not apromoter at the doses used here. Single or multipleapplications of high doses did not stimulate ornithinedecarboxybase activity. The other hyperplastic agents tested,cantharidin and ethyl phenylpropriolate, caused a slightincrease in activity, which is probably due to the weakpromoting action of these compounds (9, 15). The effect ofthese hyperplastic agents on S-Ado-Met decarboxylaseactivity, however, was much more pronounced and the timecourse of the stimulation was virtually identical to theresponse of the promoting phorbol esters. Thus the responseof this enzyme, not ornithine decarboxybase, may beassociated with the epidermal hyperplasia produced by bothpromoters and nonpromoters, while the stimulation ofornithine decarboxylase may be specific only for promoters.The peak of S-Ado-Met decarboxylase activity after treatment with a promoter or one of the hyperpbastic agentsoccurs around 12 to 18 hr, while the peak of incorporationof [3Hjthymidine into DNA after phorbol ester treatmentoccurs around 18 to 24 hr (2); the timing of these eventssuggests that increased polyamine synthesis (especiallyspermidine and spermine) may be necessary for epidermalDNA synthesis.
The effect of initiating and completely carcinogenic dosesof DMBA on ornithine decarboxylase activity is anotherindication that this response is related only to promotion(Chart 7A), although a more detailed study involving moredoses of DMBA and other carcinogens will be necessary toconfirm this point. S-Ado-Met decarboxylase activity wasalso increased after the higher dose, which may be indicativeof a hyperplastic response to this dose.
The hypothesis that elevated ornithine decarboxybaseactivity is involved in tumor promotion is supported by thepresence of very high levels of this enzyme in the 2 skincarcinomas examined and lesser increases in papiblomas(Table 1). These results agree with the consistent finding ofhigh ornithine decarboxybase activity and increased polyamine levels in malignant cells (6, 18, 24). In a spectrum ofMorris hepatomas of various growth rates, ornithine decarboxylase activity was preferentially retained or increasedwhile other pathways of ornithine utilization, as well asmany other enzymes, were decreased or lost (23). However,analysis of tumors is not a satisfactory method to determinewhich are the critical changes occurring in cells before theappearance of visible tumors. The effects of promoters thatwe observe are not the response of precancerous or initiatedcells, but of normal cells. Presumably, the specific effectsof promoter treatment would occur in all cells of skin previousby treated with an initiating dose of a carcinogen, butonly in those cells containing an initiator-caused “error―would promoter treatment confer a selective growth advantage resulting, after many promoter treatments, in tumors.The nature of the initiating error is unknown, but on thebasis of our results we would suggest that it may be asomatic mutation or a stable epigenetic change in theregulatory gene(s) controlling the level of ornithine decar
DISCUSSION
One of the problems involved in the study of themechanism of promotion has been how to distinguishbetween those biochemical effects of promoters that areessential for promotion and the many effects of thesecompounds that are nonessential. The experimental approach we have taken has been rationally to predict, basedon results from other stimulated-to-proliferate model systems, what cellular functional changes may play a role intumor promotion and to test the effect of promotertreatment on these specific biochemical processes in epidermis. If a significant change was observed compared tonormal mouse epidermis, we then examined the effect ofseveral other classes of compounds: (a) a series of structurally rebated promoters (i.e., the phorbol esters); (b) othernon-phorbol-ester promoters, with diverse chemicobiologicab properties; (c) hyperplastic agents with little or nopromoting activity; and (d) initiating and carcinogenicdosages of a polycyclic aromatic hydrocarbon, DMBA.With this systematic approach, we have asked whether theinduction of ornithine decarboxybase and S-Ado-Met decarboxylase is an essential component of promotion.
Within a series of phorbol esters of varying promotingpotencies, the response of both enzymes correlated well withtheir promoting abilities (Chart 2). Previously, a similarcorrelation was reported for graded doses of the most activeof these esters, TPA ( 14). The apparently universal abilityof promoters to cause the induction of these enzymes wasstrengthened by the results with iodoacetic acid, anthralin,and Tween 60. These compounds generally produced muchweaker responses than the phorbol esters, although the highdoses used (in the range of promoting doses, however) maybe cytotoxic to the epidermal cells.
Although the kinetics of the stimulation of ornithine andS-Ado-Met decarboxylase activities after a single application of the phorbob esters was quite different than theresponses to the other promoters tested, a significant changeoccurred when the effect of mulitple applications wasexamined. Single applications of iodoacetic acid, anthrabin,and Tween 60 generally caused a much later stimulation ofornithine decarboxylase compared to the phorbol esters;multiple applications of these compounds, however, led toan early response, which was qualitatively identical to thatobserved after single or multiple phorbol ester treatments.Thus, a characteristic effect of multiple promoter applications was a rapid, transient induction of ornithine decarboxylase activity, beginning as early as 2 hr after the lasttreatment, reaching a peak by S to 6 hr. and declining to thebasal level by 12 to 15 hr. Although S-Ado-Met decarboxybase activity was also stimulated by all the promoters tested,no common kinetic pattern after multiple applications wasobserved. The effect of repeated promoter treatments hasoften been neglected in studies on the mechanism ofpromotion; but, as we have seen with respect to ornithinedecarboxylase activity, the changes induced by multipleapplications may be qualitatively and quantitatively different from the effect of a single application.
Whereas in many tissues elevated ornithine decarboxybaseactivity (in some cases accompanied by increased S-Ado
boxylase. Such a defect would allow initiated cells tomaintain the very high enzyme levels reached after promoter treatment, while in normal cells only a transient spikeof activity occurs. After a critical number of promotertreatments, a permanently elevated level of this enzymewould be a phenotypic characteristic only of initiated cells.The sustained high level or ornithine decarboxybase activitywould lead to the accumulation of polyamines, which wouldbe the likely molecular species actually responsible for theselective growth advantage of these initiated cells.
In conclusion, we have demonstrated that the induction ofornithine decarboxylase in mouse epidermis is one of theearliest and largest effects of tumor promoters. Thisresponse is specific for promoters; all promoters tested, ofdiverse chemicobiobogical properties, were effective, whilewe have yet to find a nonpromoter that is effective in thisregard. Within a series of structurally rebated promoters(the phorbol esters), promoting ability correlates well withthe magnitude of the ornithine decarboxybase response. Theinduction of this enzyme is clearly not related to thehyperplastic property of promoters, but a good correlationwas established between S-Ado-Met decarboxylase activityand hyperplasia. The specificity of the ornithine decarboxylase response suggests that it may be useful as a short-termtest for promoting activity. Perhaps more importantly, sincea high dose of DMBA was also effective in our system, arapid in vivo screen for potential carcinogens might also befeasible.
Our knowledge of the intracellular functions of thepolyamines is very limited at this time. The availability ofspecific inhibitors of the enzymes involved in pobyaminebiosynthesis, such as methylglyoxal bis(guanybhydrazone)(25), should be helpful in defining the role of thesecompounds in both normal and malignant growth.
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