[CANCER RESEARCH 40, 308-31 5, February1980]0008-5472/80/0040-0000$02.00

Inhibition by Prostaglandin Synthesis Inhibitors of the Induction ofEpidermal Ornithine Decarboxylase Activity, the Accumulation ofProstaglandins, and Tumor Promotion Caused byI 2-O-Tetradecanoylphorbol-1 3-acetate1

Ajit K. Verma, Curtis L. Ashendel, and R. K. Boutwell2McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706

ABSTRACT

Application of i 2-O-tetradecanoylphorbol-i 3-acetate (TPA)to mouse skin results in a large and rapid induction of epidermalornithine decarboxylase (ODC; EC 4.i .i .i 7) activity, a phenotypic change proposed to be essential for skin tumor promotion. Induction of ODC activity by TPA was inhibited by priortreatment of skin with inhibitors of prostaglandin synthesis inthe order, indomethacin > naproxen > flufenamic acid > acetylsalicylic acid. In contrast, dexamethasone, a steroidal antiinflammatory drug, was ineffective at a 280-nmol dose.

The inhibitory effect of indomethacin on ODC induction wasfound to be specific. The 280-nmol dose of indomethacin thatinhibited the induction of ODC activity by 80% did not ‘inhibitthe induction by TPA of S-adenosylmethionine decarboxylaseand cyclic adenosine 3':5'-monophosphate phosphodiesterase(EC 3.i .4.17) activities. Furthermore, indomethacin treatmentaffected neither normal epidermal protein nor DNA synthesis.

The inhibition by the prostaglandin synthesis inhibitors of theinduction of ODC activity by TPA was completely overcome byconcurrent application of about 25 to 100 nmol of prostaglandins E1, E2, D2,or the 6,9-thio analog of prostaglandin 12withTPA. In contrast, prostaglandins F1,,and F2@,6-keto-prostaglandin F1@,or arachidonic acid at doses of as much as i 00nmol were ineffective. Application of 17 nmol of TPA led toabout a 3-fold increase in epidermal levels of prostaglandins Eand F, and that increase was blocked by pretreatment with 280nmol of indomethacin.

Application of 280 nmol of indomethacin before each TPAtreatment significantly inhibited the formation of skin papillomas. ProstaglandmnE2 alone could neither induce epidermalODC activity nor promote skin tumors in an initiation/promotionexperiment. However, potentiation of TPA-induced ODC activity by prostaglandin E2was observed.

The findings that the application of indomethacin prior toTPA treatment inhibits the accumulation of prostaglandins, theinduction of ODC activity, and the formation of skin papillomassuggest that (a) prostaglandins E1, E2, D2, and 12may play arole in the induction of ODC activity by TPA and that (b) TPAinduced ODC activity may be an important component of themechanism of skin tumor promotion.

INTRODUCTION

Application of the potent tumor-promoting agent TPA3 to

Received July 27, 1979; accepted November 1, 1979.I The work was supported by NIH Grants CA 071 75, CA 091 35, and CA

22484.2 To whom requests for reprints should be addressed.

3 The abbreviations used are: WA, 1 2-O-tetradecanoylphorbol-1 3-acetate

(Chemical Abstracts Registry No. 20839, 11-6); ODC, ornithine decarboxylase;

mouse skin leads to a pronounced increase (about 200-fold) inmouse epidermal ODC (EC 4.i .i .17) activity within about 5 hrafter treatment. Recent evidence indicates that this phenotypicchange is one of the essential components of the mechanismof skin tumor promotion (2, 22, 23, 34, 38).

The biochemical mechanism of induction of ODC by TPA isunclear. The role of the microtubule-containing system in regulation of ODC induction has been suggested. Thus, additionof colchicine and vinblastine in micromolar concentrations inhibits the induction of ODC activity of mouse leukemia Li 2i 0cells by fresh medium and serum (5). Similarly, i.p. administration of colchicine or other microtubule-disrupting agents, suchas vinblastine or vincnistine, prior to topical application of TPAsuppressed the induction of mouse epidermal ODC activity(24).

In most cell and tissue systems stimulated to proliferate, arapid, transient increase in ODC activity is preceded by anincrease in the level of cyclic AMP and an increase in theactivity of cyclic AMP-dependent protein kinase. Examplesinclude liver following treatment with 3-methylcholanthrene orphenobarbital and lymphocytes following treatment with mitogen (30). This relationship between an increase in cyclic AMPlevel and ODC induction was not clear in the epidermis of intactmice. (20).

In our preliminary communication (37), we reported thatindomethacin, flufenamic acid, or acetylsalicylic acid (prostaglandin synthesis inhibitors) applied to mouse skin prior toapplication of TPA remarkably depressed the induction of ODCactivity by TPA. The inhibition of the induction of ODC activityby the prostaglandmnsynthesis inhibitors was completely overcome by treatment with either PGE1 or PGE2. This suggeststhat prostaglandins may play a role in ODC induction by TPAin mouse epidermis. The role of prostaglandins in ODC induction and in skin tumor promotion by TPA is elucidated furtherin this paper.

MATERIALS AND METHODS

Materials. FemaleCharlesRiverCD-i micewere purchasedfrom Charles River Breeding Laboratories, Inc., Wilmington,Mass. and were used for experimentation at 7 to 9 weeks ofage. TPA was obtained from Dr. Peter Borchert, Eden Prairie,Minn. DMBA was purchased from Eastman Organic Chemicals,Rochester, N. Y., indomethacin was from Sigma Chemical Co.,St. Louis, Mo., and flufenamic acid and acetylsalicylic acidwere from Aldrich Chemical Co., Milwaukee, Wis. Naproxen

cyclic AMP, cyclic adenosine 3':5'-monophosphate; PG. prostaglandln; otherprostaglandins are abbreviated in the text using the basic abbreviation PG plusthe specific type, e.g., PGE is prostaglandin E.

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Prostag!andins and Epiderma! ODC !nduction

was a generous gift from Dr. Harold C. Anderson, SyntexLaboratories, Palo Alto, Calif. Prostaglandins were generouslysupplied by Dr. J. E. Pike, The Upjohn Company, Kalamazoo,Mich. DL-[i-14C]Ornithine hydrochloride (specific activity, 49.9mCi/mmol), S-adenosyl-L-[carboxy!-'4C]methionine (specificactivity, 54.6 mCi/mmol), [5,6-3H]-PGE1 (specific activity, 74Ci/mmol), [5,6-3H]PGF1a(specific activity, 74 Ci/mmol), L-[4,5-3H]Ieucine (specific activity, 50 Ci/mmol), and [methy!-3HJthymidine (specific activity, 20 Ci/mmol) were purchased fromNew England Nuclear, Boston, Mass. Rabbit antisera to PGEand PGF were purchased from Calbiochem, La Jolla, Calif.

Treatment of Mice. All mice were housed in stainless steelcages. Food and water were available ad !ibitum. Mice werekept in a normal rhythm of i 2-hr light and i 2-hr dark periods.The dorsal skin of the mice was shaved 3 to 4 days beforeexperimentation, and only those mice not exhibiting hair regrowth over this period were used. The solutions of all agentsto be applied topically were prepared in acetone and weredelivered to the shaved areas of individual mice in a volume of0.2 ml. Control mice were treated with the same volume ofacetone.

Assay of ODC and S-Adenosyl-i.-methionine DecarboxylassACtIVItY.Atappropriatetimesaftertreatment,micewerekilled by cervical dislocation, and epidermis from individualmice was separated by a brief heat treatment (i 9). ODC activityfrom soluble epidermal extracts (30,000 x g) was determinedby measuring the release of 14CO2from DL-[i -14C]ornithinehydrochloride. Assays were carried out at either i 00 @LMLornithine concentration in a final volume of 2 ml in 25-mIErlenmeyer flasks (34) or 400 @tML-ornithine in a final volumeof 0.25 ml in 15-mI Corex centrifuge tubes (36).

S-Adenosylmethionine decarboxylase activity from solubleepidermal extracts was determined by measuring the releaseof 14C02 from S-adenosyl-L-[carboxy!-14C]methionine as reported earlier (34).

Assay of Cyclic AMP Phosphodiesterase Activity. At appropriate times after treatment, mice were killed by cervicaldislocation, and epidermal homogenates were prepared. CyclicAMP phosphodiesteraseactivity in the whole epidermal homogenates was determined at 400 @Mcyclic AMP concentration (35).

Protein content of epidermal extracts was determined by theprocedure of Lowry et a!. (i 7).

Measurement of Prostaglandlns by Radiolmmunoassay.PGEand PGF levels were measured from ethyl acetate extractsof acidified (pH 2.0) mouse epidermal homogenates by radioimmunoassay as reported by Orczyk and Behrman (25). DNAcontent of the homogenates was measured by the diphenylamine method of Burton (4).

Measurement of Incorporation of Tritiated Precursors intoEpidermal Protein and DNA. Incorporation of [3H]thymidineinto epidermal DNA was determined by i.p. injection of [3H1-thymidine Ci @Ci/gbody weight) 30 mm before sacrifice asdescribed before (36). For determination of protein synthesis,mice were given [3H]leucmnei.p. (i @Ci/gbody weight) 30 mmbefore sacrifice. Epidermis from each mouse was separated bya brief heat treatment (i 9), homogenized in 2 ml of 0.5 Mperchlonc acid at 4°,and centrifuged. The pellet was washedtwice (2 x 2 ml) with ice-cold 0.5 M perchlonic acid and oncewith 100% ethanol. The pellet was dissolved in 0.5 M NaOH byheating at 80°for 30 mm. After centnifugation, aliquots of

dissolved protein were taken for determination of radioactivity.Tumor Induction Experiments. Tumors were initiatedin all

mice by application of 0.2 @imol(5i .2 @tg)of DMBA in 0.2 ml ofacetone. Beginning 2 weeks following initiation, all mice werepromoted twice a week (on Days i and 4) with either 5 (Tablei 0, Experiment i ) or 8 (Chart 6, Experiment 2) nmol of TPA forthe duration of the experiment. Mice were treated with indomethacin either 2 hr (Table 10) or 5 hr (Chart 6) before eachpromotion with TPA. Controls were pretreated with acetoneonly. There were at least 30 mice in each treatment group.Mice were housed i 0/cage in screen-bottomed stainless steelcages. The incidence of papillomas was observed weekly.

RESULTS

Effect of Treatment with Indomethacin or Other Inhibitors ofProstaglandin Synthesis on the Induction of Mouse Epidermal ODC Activity by TPA

Application of TPA to mouse skin leads to a rapid, transientinduction of ODC with peak activity at about 4.5 hr followingTPA treatment. Treatment of mouse skin with 280 nmol ofindomethacin 2 hr prior to TPA treatment results in a dramaticinhibition of the induction of ODC activity by TPA (37). The timecourse of the effect of indomethacin application relative to timeof TPA treatment on ODC induction is shown in Chart i . In thisexperiment, 280 nmol of indomethacin were applied once tothe skin at various times before and after treatment with 5 nmolof TPA, and epidermal ODC activity was determined 4.5 hrfollowing TPA treatment. Indomethacin was virtually ineffectivein inhibiting the induction of ODC activity when 24 hr wasallowed to elapse between indomethacin and TPA treatment.However, the induction of ODC activity was depressed by 79,84, and 85% when indomethacin was applied 6, 5, or 2 hrbefore TPA treatment, respectively. The induction of enzymeactivity was decreased by 52% when indomethacin was applied1 hr after TPA treatment, and the inhibition was progressively

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Chart 1. Effect of treatment with indomethacin on the induction of epidermalODC activity. Groups of mice were treated with 280 nmol of indomethacin atvarious times before and after application of 5 nmol of TPA (t 0). Mice were killed4.5 hr after TPA treatment. ODC activity from the soluble epidermal extracts wasdetermined at 100 @ML-ornithine in 2 ml final assay volume. Eachpoint representsthe mean ±S. E. of determinations of enzyme activity from soluble epidermalextracts prepared from 3 groups of mice with 3 mice/group. ODC activity inepidermal extracts prepared from mice treated with 5 nmol of TPA only was 5.4±0.67 nmol C02/30 mm/mg protein.

TIME (hr)

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Inhibition by the inhibitors of prostaglandin synthesis of the induction of ODCactivity by TPA

Mice were treated with various doses of the drugs 2 hr prior to application of5 nmol of TPA. Control mice were pretreated with acetone only. Mice were killed4.5 hr after TPA treatment, and soluble epidermal ODC activity was determinedat 400 @LML-ornithine concentration. The dose of each drug that Inhibits theinduction of ODC activity by 50% was calculated from the dose-response curveconstructed for each drug and is referred to as the median inhibitory dose in thistable.Dose

ODC activity Median inhibitoryTreatment (nmol) (% of control) dose(nmol)Acetone

100Indomethacin 280 26 110Naproxen 250 36 160Flufenamic acid 280 36 180Acetylsalicylic acid 1000 49 1100Dexamethasone 280 95 Not determined

Effect of indomethacin on incorporation of tritiated precursors intoepidermalproteinandDNAMice

were treated with either 280 nmol of indomethacin in 0.2 ml ofacetoneor0.2 ml of acetone 2 hr before treatment with acetone or 17 nmol of TPA.Micewere

killed 12 and 18 hr after TPA treatment for determination of proteinandDNAsynthesis,respectively.Mice were given i.p. tritiatedprecursor(1pCi/gbodyweight) In 0.2 ml of 0.9% NaCIsolution 30 mm before sacrifice. Eachvalueis

the mean ±S. E. of determinations from 4 groups of mice with 2mice/group.[3HJThymldlne[3HjLeucine

incorpora-incorporationTreatmenttion (dpm/@gprotein) (dpm4tgDNA)Acetone—acetone

4.49 ±0.69 77 ±6Indomethacin—acetone4.92 ±0.55 84 ±10Acetone—TPA

15.90 ±1.00 431 ±22lndomethacin—TPA14.60 ±0.5 224 ±34

A. K. Verma et a!.

with either 280 nmol of indomethacin or 1000 nmol of acetylsalicylic acid 2 hr prior to treatment with 5 nmol of TPA, andmice were killed for enzyme assay at various times followingTPA treatment (Chart 3). Application of TPA resulted in apronounced increase in ODC activity with peak activity at 6 hrfollowing TPA treatment. Enzyme activity returned to basalcontrol value at about 13 hr. Indomethacin and acetylsalicylicacid treatment resulted in a sharp reduction in the degree ofinduction of ODC activity by TPA. Furthermore, there is noindication of an altered time course of the induction of ODCactivity following inhibitor pretreatment.

Other Effects of Indomethacin Pretreatment

Is the Effect of Indomethacin on the Induction of ODCActivity Due to General Cytotoxicity? The possibility thatindomethacin may inhibit the induction of ODC activity due togeneral cytotoxicity is ruled out by a number of findings.

Application of 280 nmol of indomethacin inhibited neithernormal nor TPA-stimulated mouse epidermal protein synthesis.Furthermore, indomethacin treatment did not affect normalDNA synthesis. However, indomethacin application 2 hr priorto treatment with i 7 nmol of TPA depressed TPA-enhancedincorporation of[3H]thymidmneinto DNA (Table 2). These resultsare in accord with previous findings (7, 8).

4TIME AFTER TREATMENT WITH TPA (hr)

less as the interval between the time of its application and timeof assay was shortened further. Thus, when indomethacin wasapplied 2.5 hr after TPA treatment, the inhibition was only39%.

Inhibition of the induction of ODC activity by indomethacinwas dose dependent (Chart 2). Inhibition of enzyme inductionwas observed at a dose above 25 nmol. Application of i 50 and300 nmol of indomethacin 2 hr before treatment with 5 nmol of

TPA inhibited the induction of ODC activity by 54 and 71%,respectively.

A number of other nonsteroidal antiinflammatory drugs whichare known to inhibit prostaglandin synthesis were tested fortheir ability to inhibit the induction of ODC activity, and the dataare summarized in Table i . The relative inhibitory potency wasin the order, indomethacin > naproxen > flufenamic acid >acetylsalicylic acid. A single application of 280 nmol of dexamethasone, a steroidal antiinflammatory drug, did not inhibitthe induction of ODC activity when applied in 0.2 ml of acetone2 hr before TPA treatment. However, a detailed dose- and timeresponse relationship was not determined.

The possibility that treatment with prostaglandmnsynthesisinhibitors may alter the time course of induction of ODC activityby TPA was explored. In this experiment, mice were treated

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DOSE OF INDOMETHACIN (nmol)

Chart 2. Effect of indomethacin dose on the induction of epidermal 0DC@activity. Mice were treated with various doses of indomethacin 2 hr beforeapplication of 5 nmol of TPA. Mice were killed for ODC assay 4.5 hr after TPAtreatment. Soluble epidermal ODC activity was determined at 400 @a@iL-ornithineconcentration. Each point represents the mean of duplicate determinations ofenzyme activity from soluble epidermal extracts prepared from 4 mice.

Table 1

Chart 3. Effect of indomethacin or acetylsalicylic acid pretreatment on theinduction of ODC activity by TPA. Mice were treated with acetone (•),280 nmolof indomethacin (0), or 1000 nmol of acetylsalicylic acid (A) 2 hr prior totreatment with 5 nmol of TPA. Mice were killed at the Indicated times followingTPA treatment. ODC activity from soluble epidermal extracts was determined at400 @ML-omithine concentration. Each point is the mean of duplicate determinations of enzyme activity from soluble epidermal extracts prepared from 4 mice.

Table 2

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0DCactivityinmixedsolubleepidermalextractspreparedfromacetone-orTPA-treatedmouse epidermis pretreated with either acetone orindomethacinMice

were treated with 280 nmol of indomethacin 2 hr before application of5nmolof TPA. Control mice were treated with acetone. Mice were killed 4.5hrafterTPA treatment. Soluble epidermal extracts were prepared, and ODCactivityin

extracts was determined. Each value is the mean of duplicatedeterminationsofenzyme activity from 3 groups of mice. Each group contained thecombinedextracts

from 3 mice.ODC

activityTreatment(nmol CO2/30mm)Experiment

1A.Indomethacin + acetone0.00B.Indomethacin+ TPA0.16C.Acetone + TPA0.50A+CCalculated

0.50Experimental0.54B+CCalculated

0.66Experimental0.73Experiment

2A.Acetone + TPA0.49B.Indomethacin+ TPA0.02A+BCalculated

0.51Experimental0.67

Effect ofpretreatment with indomethacin on the induction of ODC andS-adenosylmethionine decarboxylaseactivitiesMice

were treated with acetone or 280 nmol of indomethacin 2 hr prior totreatment with acetone or 5 nmol of TPA. Mice were killed for ODC and Sadenosylmethlonlne decarboxylase assays 4.5 and 24 hr after TPA treatment,respectively. OD@activity from the soluble epidermal extracts was measured at100 @*ML-OrfllthlfleconcentrationIn a final assay volumeof 2 ml. Each valuerepresents the mean ±S. E. of determination of enzyme activity from 3 groupsof mice. Each group contained the combined supernatants prepared from 3mice.(nmol

C02/30 mm/mgprotein)Treatment

ODC activity SAMD°activityAcetone

+ acetone 0.02 0.04 ±0.01Acetone+TPA 3.14±0.48 0.20± 0.02Indomethacin + WA 0.59 ±0.05 0.28 ±0.03a

SAMD. S-adenosylmethlonmne decarboxylase.

Effect of indomethacin treatment on the induction of cyclicAMPphosphodiesteraseactivityMice

were treated with acetone or 280 nmol of indomethacin 2 hrbeforeapplicationof acetone or 10 nmol of TPA. Mice were killed 6 hr afterTPAtreatment.

Low-affinity cyclic AMP phosphodlesterase activity in wholeepidermalhomogenatesat 400 ga@isubstrate concentration was determined. Each valueisthe

mean ±S. E. of determination of enzyme activity in wholehomogenatespreparedfrom 5 Individual mice.Cyclic

AMPphosphodiesteraseTreatmentactivity (pmol/mmn/mgprotein)Experiment

1Acetone+ acetone 292 ±6Acetone+ TPA 883 ±85Indomethacin

+ TPA 850 ±105ExperIment

2Acetone+ acetone 281 ±35Acetone+ TPA 906 ±78Indomethacin

+ TPA 1089 ±137

Table6Effectof pretreatment with prostaglandin synthesis inhibitors on theinductionof

mouse epidermal ODCactivityMicewere treated with either acetone or 280 nmol of theprostaglandmnsynthesisinhibitor in acetone 2 hr before application of either 10 nmol of TPAor1

0 nmol of TPA containing 70 nmol of PGE2.Mice were killed 4.5 hr laterforODCassay at 100@ L-omithine concentration. Each value is the mean ±S.E.of

determinations carried out on 3 groups of mice with 3mice/group.ODC

activity (nmol CO2/30 mm/mgprotein)Treatment

—PGE2 +PGE2Acetone

4.1 ±0.68 8.06 ±1.19Indomethacin0.85 ±0.10 6.18 ±0.58Naproxen1.75 ±0.10 6.02 ±0.45Flufenamic

acid 1.92 ±0.24 5.09 ±0.65Acetylsalicylicacid 2.52 ±0.24 5.71 ±0.62

Prostag!andins and Epiderma! ODC Induction

The dose of indomethacin that dramatically inhibited theinduction of ODC activity failed to inhibit the induction of Sadenosylmethionine decarboxylase activity by TPA (Table 3).

Addition of as much as 560 nmol of indomethacin to solubleepidermal homogenates prepared 4.5 hr after TPA treatmentof mouse skin did not alter enzyme activity when assayedunder normal assay conditions (ODC activity was 3.84 nmol/30 mm/mg protein in the absence and was 3.99 nmol/30 min/mg protein in the presence of 560 nmol of indomethacin).

Effect of Indomethacin Pretreatment on TPA-InducedCyclic AMP Phosphodiesterase Activity. Indomethacin inhibits prostaglandin synthesis by irreversibly inactivating cyclooxygenase (6). In addition, indomethacin has been shownto inhibit a number of enzymes including cyclic AMP phosphodiesterase. However, a higher concentration of indomethacinis required for the latter effects (6). The possibility that indomethacin treatment may inhibit the induction of cyclic AMPphosphodiesterase activity by TPA was examined, and theresults are shown in Table 4. Application of i 0 nmol of TPA ledto a 3-fold increase in cyclic AMP phosphodiesterase activitymeasured at 6 hr following TPA treatment in accord with ourprevious findings (35). Treatment with 280 nmol of indomethacm 2 hr before TPA treatment did not affect the degree ofenzyme induction (Table 4). In contrast, epidermal ODC activitywas inhibited by 80% following indomethacin pretreatment(Table 3).

Does Indomethacin Pretreatment Lead to the Productionof an Inhibitor of TPA-Induced Activity? Mixing of epidermal

Table 3

extracts prepared from acetone- and indomethacin-pretreatedepidermis resulted in simple additive ODC activity (Table 5);this suggests that indomethacin treatment does not lead to theproduction of inhibitor(s) of TPA-induced ODC activity.

Effects of Prostaglandinson Indomethacin-causedInhibitionof the Induction of Epidermal ODC Activity by TPA

Inhibition of the induction of ODC activity by indomethacincan be completely overcome by concurrent application ofeither PGE1or PGE2with TPA. Counteraction of the ir@domethacm inhibition was dependent on the dose of prostaglandmns.Application of 70 nmol of PGE2completely counteracted inhibition of the induction of ODC activity by 280 nmol of indomethacin (37). As shown in Table 6, application of 70 nmol ofPGE2 concurrently with TPA also completely counteractedinhibition of the induction of ODC activity by various prostaglandin inhibitors. As reported earlier (37), application of asmuch as 70 nmol of PGE2alone did not induce epidermal ODCactivity. In contrast, PGE2 applied with TPA potentiated theinduction of ODC activity by TPA. The time course of the effectof application of PGE2alone or concurrently with TPA to normal

Table 5

Table 4

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Table7Effectof prostaglandins on inhibition of the induction of ODCactivitybyindomethacinGroups

of mice were treated with either acetone or 280 nmol ofIndomethacininacetone 2 hr before treatment with either 5 nmol of TPA or 5 nmol ofTPAcontaining

various doses of prostaglandmn.Mice were killed 4.5 hr after thelasttreatment.ODCactivity from the soluble epidermal extracts was measured at400@LM

L-Omlthine concentration. Each value represents the mean ± SE. of determinations carried out on 3 groups of mice with 3mIce/group.ODC

activity (nmol C02/30 mm/mgprotein)Dose

Pretreated with PretreatedwithCompound(nmol) acetoneindomethacinNone

7.00 ±1.29 2.21 ±0.24PGE225 8.51 ±1.36 4.09 ±0.71100

8.63 ±1.26 7.43 ±1.43POD225 6.93 ±0.55 3.03 ±0.28100

6.96 ±0.73 5.97 ±0.676,9-Thio-PGI225 8. 16 ±0.34 5.43 ±1.00100

8.55 ±1.63 8.22 ±1.766-Keto-PGF,,25 6.72 ±0.94 2.01 ±0.27100

7.12 ±0.09 3.30 ±0.31Arachidonicacid 100 7.72 ±1.07 2.18 ±0.06

A. K. Verma et a!.

or indomethacin-pretreated mouse skin on epidermal ODCactivity is shown in Chart 4. Inhibition of the induction of ODCactivity by indomethacin was completely counteracted by application of 70 nmol of PGE2concurrently with TPA. Applicationof PGE2 alone did not induce PDC activity at any of the timepoints (Chart 4) or doses (Chart 4, inset) studied.

A number of other prostaglandmns(Chart 5) were tested fortheir ability to overcome inhibition of ODC induction by indomethacin, and the data are summarized in Table 7. Applicationof 25 or i 00 nmol of PGE2,PGD2,or 6,9-thio-PGI2 completelyovercame the inhibition of ODC induction by indomethacin. Incontrast, i 00 nmol of 6-keto-PGF1,,or arachidonic acid wereineffective (Table 7). PGF1,,or PGF20was also unable to counteract indomethacin-caused inhibition of the induction of ODCactivity (37).

Does PGE2Counteract RetinoicAcid-causedInhibitionof theInduction of ODC Activity?

Retinoic acid is a potent inhibitor of the induction of mouseepidermal ODC activity by TPA (34). It was of interest todetermine whether PGE2 could also counteract the inhibitionof the induction of ODC activity by retinoic acid. As shown inTable 8, application of 0.i 7 nmol of retinoic acid 2 hr prior toTPA treatment inhibited by 85% TPA-induced ODC activity.Application of PGE2concurrently with TPA failed to counteractenzyme inhibition caused by retinoic acid. Furthermore, simultaneous application of 280 nmol of indomethacin and 0.17nmol of retinoic acid to mouse skin resulted in inhibition of theinduction of ODC activity greater than that obtained with retinoic acid on indomethacin alone. These results suggest thatinhibition of the induction of ODC activity by retinoic acid maynot involve inhibition of synthesis of prostaglandins.

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Chart 4. Effect of PGE2on indomethacin-caused inhibition of the induction ofODC activity. Groups of mice were treated with acetone (•)or 280 nmol ofindomethacin (O—O) 2 hr before application of 5 nmol ofTPA. PGE2,70 nmol,was applied concurrently with TPA to indomethacin-pretreated mouse skin(0- - -0). PGE2,70 nmol,alone(A)wasapplied.Inset,variousdosesof POE2applied alone (A) or with 1.7 nmol of TPA (C). Mice were killed 4.5 hr after lasttreatment. ODC activity from soluble epidermal extracts was determined at 400@LML-Ornithineconcentration.Eachpointisthemeanofdeterminationsofenzymeactivity from 2 groups of mice with 3 mice/group.

I—OXIDATION

@‘\ PI4OSPHOLIPID\\\ @PHOSPHOLIPASE62

•LIPOXYGENASE

ARACHIDONIC ACID

CYCLO-OXYGENASE

Hoo\@@::I::IIT_CooHHETE

Th@OM@ISYNTHETASE

OHTHROMBOXANE62

PROSTACYCLI4(PGI@) OH 1HYDROLYSIS

tMILDACID.t:_L:I.::::c::::.::@...@ ThRO1I@BOXANES@

HO 0

6H

6 KETO PGFC

HO

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PGF@

PROSTAGLANDINBIOSYNTHESIS

Chart 5. Prostaglandin biosynthesis. HHT, 12-i.-hydroxy-5,8, 10-heptadecatrienoic acid; HETE, 12-L-hydroxy-5,8, 10,14-eicosatetraenoic acid.

Effect of Indomethacin Pretreatment on Accumulation ofProstaglandins by TPA

The possibility that indomethacin may inhibit the induction ofODC activity by inhibiting the accumulation of prostaglandinswas examined, and the results are shown in Table 9. In thisexperiment, mice were treated with either acetone or 280 nmolof indomethacin 2 hr prior to application of i 7 nmol TPA. Micewere killed at various times following TPA treatment, and PGEor PGF was quantitated by radioimmunoassay. A 2-fold increase in PGE levels was detected as early as 4 hr followingTPA treatment, and a peak level was observed between 6 andi 4 hr. PGF levels were also increased at 4, 6, and i 4 hr afterTPA treatment. Indomethacin pretreatment completely in

4 8 2 6 20TIME AFTER TREATMENT WITH TPA (hr)

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Effect of POE2on indomethacin- or retinoic acid-caused inhibition oftheinductionof ODCactivityGroups

of mice were treated with acetone, 280 nmol of Indomethacin, or0.17nmolof retinoic acid 2 hr prior to treatment with 5 nmol of TPA or 5 nmol ofTPAcontaining

70 or 140 nmol of POE2. Mice were killed 4.5 hr after thelasttreatment,and soluble epidermal ODC activity was measured at 400 llM L

ornlthlne concentration. Eachvalue represents the mean ±SE. ofdeterminationscarriedout on 3 groups of mice with 3 mice/group.ODC

activity(nmolCO2/30 min/ %ofTreatment

mg protein)controlAcetone

+ acetone0.02Acetone+ WA 5.81 ±0. 15100Indomethacin

+ TPA 1.38 ±0.4124Indomethacin+ TPA + pGE2a 6.37 ±0.78110Retinoic

acid + WA 0.85 ±0.0815Retinolcacld+TPA+PGE2a1.18±0.14 20

Retinoic acid + ‘IRA+ PGE2b 0.85 ±0.1415Indomethacin+ retinoic acid + TPA 0. 14 ±0.08 2

Treatment(nmol

CO2/30 mm/mg protein)Papillomas/

mouse%of papillo

masSAMDaactiv

ODC activityityAcetone

+ acetoneAcetone + TPAIndomethacin + TPA0.02

0.04 ±0.016.76 ±0.88 0.25 ±0.051.80 ±0.57 0.31 ±0.030.00

12.16.10.00

9084

Table9Effectof indomethacin on TPA-stimulated epidermal PGE and PGFlevelsGroupsof mice were treated with either 0.2 ml of acetone or 280 nmolofindomethacin

In 0.2 ml of acetone 2 hr prior to application of 17 nmol ofTPA.Micewere killed at the indicated times. Epidermal POEand PGFwerequantitatedby

radloimmunoassay (see ‘Materialsand Methods―).Each value is the meanof4determinatIonscarriedout on 4 mice(variation,<15%).(pmol/mg

DNA)Treatment

Time (hr) PGEPGFNone

104Acetone

+ WA 4 22.96.4636.78.31435.921.4Indomethacin

+ TPA 4 15.14.4612.74.51414.2 1.7

Prostag!andins and Epiderma! ODC !nduction

Table 8 ind@methacin treatment on the formation of skin papillomaswas determined, and the results are shown in Chart 6. Thenumber of papillomas per mouse was significantly decreasedfollowing indomethacin pretreatments, but the effect on tumorincidence was small. The inhibition of formation of skin papillomas was not due to toxicity. The average weight of micetreated with indomethacin did not differ from that of controls.Repeated applications of indomethacin at a 560-nmol dosewere toxic. Similar results have been observed by others (39).

Table 10Effect of indomethacin pretreatment on the induction of ODC and

S-adenosylmethionine decarboxylase activities and formation of skin papillomasAll mice were initiated with 0.2 @molof DMBA in 0.2 ml of acetone. Two weeks

after initiation, mice were treated twice a week with either 0.2 ml of acetone or280 nmol of indomethacin in 0.2 ml of acetone 2 hr before each application of 5nmol of TPA for the tumor induction experiment and of 8 nmol of TPA fordetermination of ODC and S-adenosylmethionine decarboxylase activities. Micewere killed 4.5 and 18 hr after the 11th application of TPA for determination ofODC and S-adenosylmethionmnedecarboxylase activities, respectively. Eachvalue represents the mean ±S. E. of determinations of enzyme activities from 6individual mice. Incidence of skin papillomas was recorded after the 20th weekof promotion, and there were 30 mice in each treatment group.

a 70 nmol PGE2.

b 140 nmol POE2.

a SAMD, S-adenosylmethionine decarboxylase.

WEEKS OF PROMOTION

Chart 6. Effect of indomethacin treatment on formation of skin papillomas. Allmice were initiated with 0.2 @&molof DMBA and 2 weeks later were treated twicea week with 0.2 ml of acetone (•),28 nmol of indomethacin (A), or 280 nmol ofindomethacin (0) in 0.2 ml of acetone 5 hr before application of 8 nmol of TPAfor the duration of the experiment. There were 30 mice/cage, and the incidenceof papillomas was observed weekly.

hibited the increased accumulation of prostaglandins at all ofthe time points examined. Recently, a more detailed timecourse of the effect of tumor-promoting phorbol esters on PGEand PGF levels in mouse epidermis has been reported (i).

Effect of Indomethacin on Formation of Skin Papillomas

In order to establish further the correlation between inductionof epidermal ODC activity and skin tumor promotion by TPA,the effect of indomethacin treatment on the formation of skinpapillomas was investigated, and the results of the first experiment are summarized in Table i 0. In this experiment, micewere initiated with 0.2 @tmolof DMBA and 2 weeks later weretreated twice weekly with either acetone or 280 nmol of indomethacin 2 hr before each application of 8 and 5 nmol of TPAfor determination of enzyme activities and the induction of skintumors,respectively.Groupsofmicethatweretreatedwith8nmol of TPA twice a week were killed for enzyme assays afterthe 11th application of TPA, while the other groups of micewhich received 5 nmol of TPA twice a week were continuouslytreated, and tumor incidence was observed after the 20th weekof promotion. The dose of indomethacin (280 nmol) that inhibited by 73% the induction of ODC activity measured 4.5 hrafter the 11th application of TPA inhibited by 50% the numberof skin papillomas recorded after the 20th week of promotion(Table 10). In a separate experiment, a more detailed effect of

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A. K. Verma et a!.

9). Accumulation of PGE by TPA could be blocked by pretreatment with indomethacin, suggesting that inhibition of the induction of ODC activity by indomethacin may involve the inhibition of an increase in PGE levels by TPA that may play a roleinODCinduction.However,adetailedanalysisofaccumulationof all prostaglandmns(Chart 5) following TPA treatment was notobtained. Furthermore, it remains to be determined in mouseepidermis whether enhanced PGE levels are specific for promoters (i ) or can be increased following nonpromoting hyperplastic agents and whether the antipromoting activity of retinoicacid (38) and dexamethasone (39) involve their ability to affectTPA-enhanced accumulation of prostaglandins.

Although the data suggest that prostaglandins may mediateODC induction by TPA in mouse epidermis, application of thoseprostaglandins alone, which counteracted indomethacin inhibition, did not induce epidermal ODC activity. However, insome experiments, application of certain prostaglandins withTPA potentiated the induction of ODC activity by TPA (Chart4). These findings suggest that prostaglandmnsmay be necessary but are not sufficient for ODC induction by TPA. TPA mustrelease some other factors in addition to prostaglandins whichare important for induction of ODC activity by TPA. However,addition of PGE2alone has been shown to induce ODC activityin cultured cells (26).

Application of PGE2 alone could not promote skin tumorformation in an initiation/promotion experiment (data notshown). In contrast, Lupulescu (i 8) reported that PGE2whengiven i.m. concomitantly with 3-methylcholanthrene 3 times aweek enhanced formation of skin carcinomas in male albinoSwiss mice.

The data showing that prostaglandins alone can neitherinduce ODC activity nor promote skin tumor formation implythat the relationship between prostaglandmnsand promotion iscomplex. However, the findings that indomethacin treatmentbefore application of TPA inhibits the accumulation of prostaglandmns,the induction of ODC activity, and the formation ofskin papillomas strongly suggest that prostaglandins play arole in ODC induction as well as skin tumor promotion. Theexact biological role of prostaglandins during skin tumor promotion remains speculative.

ACKNOWLEDGMENTS

We thank Hope Rice, Barbara Shapas, and Tim O'NelI for expert technicalassistance as well as The Upjohn Company for a generous supply of prostaglandma.

REFERENCES

1. Ashendel, C. L, and Boutwell, R. K. Prostaglandin E and F levels in mouseepidermis are increased by tumor-promoting phorbol esters. Blochem. Biophys. Res. Commun., 90: 623-627, 1979.

2. Boutwell, R. K. The role of the Induction of ornithine decarboxylase activityin tumor promotion. In: H. H. Hiatt, J. D. Watson, and J. A. Winsten (eds.),Origins of HumanCancer, Book B, pp. 773—783.Cold Spring Harbor, N. V.:Cold Spring Harbor Laboratory, 1977.

3. Brune, K., Kahn, H., Schmidt, K., and Hecker, E. Inflammatory, tumorinitiating and promoting actMtles of polycyclic aromatic hydrocarbons andditerpene esters of polycyclic aromatic hydrocarbons and diterpene estersin mouse skin as compared wIth their prostaglandln releasing potency invitro. Cancer Left., 4: 333-342, 1978.

4. Burton,K. A. Studyof the conditionsand mechanismof the diphenylaminereaction for the colorlmetric estimation of deoxyribonucleic acid. Biochem.J., 62: 315—323,1956.

3i 4 CANCERRESEARCHVOL. 40

DISCUSSION

Prostaglandins are naturally occurring cyclic metabolites ofunsaturated fatty acids that have numerous physiological functions (i 0, 27, 28, 3i , 32). A number of prostaglandins intissues of various species are biosynthesized from their precursor, unsaturated fatty acids present in cell membranes inthe form of phospholipids (Chart 5). Biosynthesis of PGE2andPGF2from the precursor, arachidonic acid, has been shown inthe skin of various species including mice and humans. Theprostaglandin synthetase is mostly microsomal (i 4, 4i ) and ispresent in the epidermis (40). Prostaglandmnsare implicated asmediators of cutaneous inflammation as well as various pathological skin conditions (9, i 0, 28). Injections of PGE2 andPGF20appeared to enhance formation of squamous cell carcinoma in mice treated with 3-methylcholanthrene (18). Elevatedlevels of prostaglandins are found in various tumors (i 3).Addition of carcinogens or tumor-promoting phorbol diestersto cultured dog kidney cells (MDCK) stimulates the release ofprostaglandmnsin the medium (i 1, 12, 15, 16), and a correlationhasbeenshownto existbetweenirritantandpromotingactivity in mouse skin and PGE2release by macrophages by aseries of tumor promoters (3). Furthermore, tumor promoterscause an enhanced synthesis of epidermal phospholipids (29)and accumulation of PGE and PGF levels, and the activities ofvarious phorbol esters for increasing PGE levels paralleledtheir tumor-promoting activity (i ). Now, we report that prostaglandins may play a crucial role in TPA-induced ODC activity,an enzyme involved in mouse skin tumor promotion (2, 22, 38).

The results presented indicate that mouse skin treated withindomethacin, naproxen, flufenamic acid, or acetylsalicylic acid(prostaglandin synthesis inhibitors) before TPA treatment inhibited the induction of epidermal ODC activity (Table i ). It ishighly unlikely that the inhibitory effect of these agents on

enzyme induction is the result of general cytotoxicity. Application of indomethacin affected neither normal epidermal protein nor DNA synthesis (Table 2), nor did it inhibit the inductionof S-adenosylmethionine decarboxylase (Table 3) or cyclicAMP phosphodiesterase (Table 4), the other enzymes knownto be induced by TPA treatment to mouse skin (2i , 23, 35). Itis more probable that the'suppression of enzyme induction bythe nonsteroidal antiinflammatory agents is the result of eithera direct inhibition of prostaglandin synthetase or the release ofprostaglandins (6, 33). Interestingly, the ability of prostaglandmnsynthetase inhibitors to inhibit the induction of ODC activitycorrelates with their ability to inhibit prostaglandin synthetasein in vitro systems (6). However, we did not measure the activityof prostaglandin synthetase in mouse epidermis.

Convincing evidence strengthening the role of prostaglandins in ODC induction is the observation that the inhibition ofthe induction of ODC activity by the inhibitors of prostaglandmnsynthesis was overcome by application of prostaglandins concurrently with TPA. PGE1,PGE2,PGD2,and 6,9-thio-PGI2 wereeffective whereas PGF1a, PGF2a, 6-keto-PGF1a, and arachidonic acid were inactive in their ability to counteract the inhibition of the induction of ODC activity by indomethacin (Table7; Ref. 37). These results suggest that indomethacin mayinhibit the production of those prostaglandmnsthat modulateODC induction by TPA. This possibility was strengthened bythe finding that TPA treatment led to an enhanced accumulationof PGE levels as early as 4 hr following TPA treatment (Table

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Prostaglandins and Epidermal ODC Induction

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biosynthesis by vasoactive substances in methylcholanthrene-transformedmouse Balb/3T3. J. Biol. Chem., 257. 776-780. 1976.

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13. Jaffe, B. M. Prostaglandins and cancer—an update. Prostaglandins, 6:453-461, 1974.

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albino Swiss mice. J. Nati. Cancer Inst., 67: 97-106, 1978.19. Marrs, J. M., and Voorhees, J. J. A method for bioassay for an epidermal

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21. Mufson, R. A., Simsiman, R. C., and Boutwell, R. K. Increased cyclicadenosine 3':5'-monophosphate phosphodiesterase activity in the epidermisof phorbol ester-treated mouse skin and in papillomas. Cancer Res., 39:2036-2040. 1979.

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1980;40:308-315. Cancer Res   Ajit K. Verma, Curtis L. Ashendel and R. K. Boutwell  by 12-O-Tetradecanoylphorbol-13-acetateAccumulation of Prostaglandins, and Tumor Promotion Causedof Epidermal Ornithine Decarboxylase Activity, the Inhibition by Prostaglandin Synthesis Inhibitors of the Induction

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