[CANCER RESEARCH 43,4126-4131, September 1983]

Effects of Single Applications of 12-O-Tetradecanoylphorbol-13-

acetate, Mezerein, or Ethylphenylpropiolate on DMA Synthesis

and Polyamine Levels in Hairless Mouse Epidermis

Jan Erik Paulsen1 and Erie Grieg Astrup2

Institute of Pathology, University of Oslo, Rikshospitalet, The National Hospital, Osto 7, Norway

ABSTRACT

Quantitative and qualitative changes in epidermal polyaminelevels and DNA synthesis (specific activity and labeling index)after a single topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA), mezerein (MEZ), or ethylphenylpropiolate

(EPP) in acetone were studied concomitantly in the same epidermal cell population from each treated mouse. The dosestested were 17 nmol of TPA; 1.7, 8.5, and 17 nmol of MEZ; and20 i<mol of EPP. With relatively small variations in time patterns,both the tumor promoter TPA and the mitogens MEZ and EPPcaused similar sequential changes: initial inhibition of DNA synthesis; induction of L-omithine decarboxylase activity; and sub

sequent peaks of putrescine levels preceding peaks in the rateof DNA synthesis. A remarkably good correlation between themolar ratio of spermidine/spermine and the increase in DNAsynthesis was seen after all three of the compounds. However,in the initial period with inhibited DNA synthesis, a negativecorrelation between spermidine/spermine and DNA synthesiswas observed after all of the treatments. TPA and MEZ inducedpronounced biphasic increases in DNA synthesis, accumulationof putrescine, and the spermidine/spermine ratio, whereas EPPinduced single-peaked increases in the same variables. The

fluctuations in polyamine levels and DNA synthesis were associated with cohorts of partly synchronized cells passing the cellcycle multiple turns. Thus, the induction of L-omithine decarbox

ylase and the polyamines does not seem to be specific for tumorpromotion but merely seems to be associated with the cell kineticevents during stimulated cell proliferation. It is suggested thatquantitative aspects of hyperprdiferation may be essential fortumor promotion.

INTRODUCTION

A difficult problem in 2-stage carcinogenesis is to determine

the specificity of promotion (7,23) by trying to separate putativespecific effects of strong tumor promoters from those of othermitogens. Several responses have, in turn, been associated withtumor promotion; induction of ODC3 and the polyamines (7),

reduction of /3-adrenergic accumulation of cyclic AMP (24), increased protease activity (37), increased induction of plasmino-gen activator (40), and increased number of so-called dark cells

(19). However, various experiments indicate lack of specificityfor some of these responses. Takigawa ef al. (35) showed thatEPP, a good hyperplasiogenic agent but a weak tumor promoter

1To whom requests for reprints should be addressed.2Supported by the Norwegian Society for Fighting Cancer.3The abbreviations used are: ODC, L-omithine decarboxylase (EC 4.1.1.17);

EPP, ethylphenylpropiolate; TPA, 12-O-tetradecanoylphorbol-13-acetate; MEZ,mezerein; HPLC, high-performance liquid chromatography; LI, labeling index.

Received November 2,1982; accepted June 6,1983.

(26), induces ODC and polyamines. Marks et al. (22) demonstrated that ^-O-tetradeca^-c/s^-frans-e.S-tetradecanoylphor-bol-13-acetate, a weak promoter with hyperplasiogenic effects,

induces ODC and polyamines like TPA, which is the strongesttumor promoter known. Mufson et al. (25) showed that MEZ, aweak tumor promoter (25, 32), induces epidermal ODC activity,hyperplasia, and reduction of /3-adrenergic accumulation of cyclic

AMP to the same extent as does TPA.Generally, the polyamines seem to be associated with cell

proliferation, DNA synthesis, RNA synthesis, protein synthesis,cell differentiation, and a number of other cell functions (4, 13,14, 18). We have previously reported the relationship betweenalterations in epidermal polyamine levels and DNA synthesisafter a single application of TPA (4). In accordance with a cellkinetic study of TPA-induced epidermal effects (2), we showed

(4) that the molar ratio of spermidine to spermine correlated wellwith the waves of DNA synthesis and number of cells in S phase,and peaks of putrescine were seen previous to the increases ofspermidine/spermine ratio and DNA synthesis.

Though induction of polyamines may not be specific to tumorpromotion, the polyamines may be critically involved, as thefollowing reports indicate: retinoids inhibit ODC activity, polyamine accumulation, and TPA-induced papilloma formation (38);the irreversible ODC inhibitor a-difluoromethylomithine inhibitsTPA-induced papilloma formation (33, 36, 39); putrescine giveni.p. enhances TPA-induced tumorigenesis (39); and the putrescine level in TPA-induced papillomas is considerably higher than

that of surrounding skin tissue (39). However, the significance ofquantitative and qualitative changes in polyamine synthesis withrespect to tumor promotion has only sparsely been investigated(39). The aim of this study is: (a) to establish equal experimentalconditions in order to compare the effects of the tumor promoterTPA with the corresponding effects of the weak or non-tumorpromoters MEZ and EPP on hairless mouse epidermis; (b) tocompare quantitative and qualitative patterns of epidermal DNAsynthesis and polyamine synthesis after these 3 treatments; and(c) to demonstrate in more detail the relationship between polyamine levels and DNA synthesis.

MATERIALS AND METHODS

Materials. Hairless mice (hr/hr Oslo strain) of both sexes, 8 to 9weeks old (17), were used. At each time point studied, the animals usedwere of the same sex. TPA and MEZ were obtained from Dr. PeterBorchert (University of Minnesota, Minneapolis, Minn.), and EPP waspurchased from Aldrich Europe, Beerse, Belgium. oL-[1-14C]Omithinehydrochloride (52.8 mCi/mmol) and [mef/iy/-3H]thymidine (5.0 Ci/mmol)

were from Radiochemical Centre, Amersham, United Kingdom. Calfthymus DNA, dimethylaminonaphthalenesulfonyl (dansyl) chloride, L-pro-line, 1.6-diaminohexane. and the hydrochloride salts of putrescine, sper-

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Epidermal DNA and Polyamine Synthesis after TPA, MEZ, or EPP

midine, and spermine used as standards were from Sigma Chemical Co.,St. Louis, Mo. Methanol, HPLC grade, was from Rathbum ChemicalsLtd., Walkerburn, Peeblesshire, Scotland. The HPLC equipment wasfrom Waters Associates, Milford, Mass.

Treatment of Mice. Animals were housed in plastic cages in a roomwith a 12-hr light-dark rhythm and controlled temperature and humidity;

they were given a standard diet and water ad libitum. The cages werecleaned and fresh water was supplied at noon 3 times a week. 17 nmolof TPA, 20 ^mol of EPP, and 1.7, 8.5, and 17.0 nmol of MEZ, allcompounds dissolved in 0.2 ml of reagent-grade acetone, respectively,

were delivered to the dorsal skin of the animals. Control mice weretreated with the same volume of reagent-grade acetone. Because of the

diurnal rhythms of epidermal cell proliferation (12), animals were alwayskilled between 1 and 2 p.m.

Determination of Polyamines. The epidermises from 2 mice wereused for each sample analyzed. The mice were killed by cervical fracture,the skins were excised, and the epidermises were isolated and homogenized as described previously (1). Polyamines were extracted in 2 ml of0.2 N perchloric acid. After centrifugation, 200 ¡Aof the supernatant weremixed with 50 n\ of 1.6-diaminohexane (internal standard, 215 nmol/ml)and 100 /il of saturated Na2CO3. Dansylation with dimethylamino-

naphthalenesulfonyl (dansyl) chloride (30 mg/ml of acetone) was performed as described (31 ). The reaction mixture was kept for 10 hr in thedark at room temperature. Excess dansyl chloride was removed byadding 100 n\ of proline (150 mg/ml, 30 min). Dansyl derivatives wereextracted into 500 n\ of toluene by vigorous shaking. Then 200 n\ of thetoluene fraction were evaporated at 60°,and the residue was resolved

in 100 n\ of methanol before injection of 15 /¿Ion the HPLC column. Theexternal standard, prepared as described above, contained 10.8 nmol of1,6-diaminohexane, 5 nmol of putrescine, 10 nmol of spermine, and 25

nmol of spermidine in 250 ^l of 0.2 Nperchloric acid. The HPLC procedure(described by Seiler and Knödgen; Ref. 29) was used with minor modifications. Separation of dansyl derivatives was achieved on a reversed-

phase column (Rad Pack A, 3.9 x 300 mm, Waters Associates) using agradient with 65% methanol as Solvent B and 100% methanol as SolventA. The gradient changed from 100% of Solvent B to 100% of Solvent Ain 23 min at a flow rate of 2 ml/min. All amines were eluted within 23min. The column was allowed to reequilibrate with Solvent B for 5 minbefore the next run. The dansyl derivatives were quantified using afluorescence detector. An excitation filter at 360 nm and a 460 nm cutoff filter for the emitted light were used.

Specific Activity. For pulse-labeling of DNA, 30 »Ciof [methyl-3»]

thymidine dissolved in 0.3 ml of 0.9% NaCI solution were injected i.p. 30min prior to killing of the animals. Following extraction of the polyaminesfrom the homogenized, pooled epidermis, the pellet was washed twicewith 2.5 ml of ice-cold 0.2 N perchloric acid and once with 4 ml of ethanol/ether (1/1, v/v) to remove the acid-soluble nucleotides. DNA in the pelletwas then hydrolyzed in 0.5 N perchloric acid at 90°for 10 min. Following

centrifugation, duplicate aliquots of hydrolyzed DNA (0.3 ml) were determined for radioactivity in a Packard Tri-Carb liquid scintillator spectrom

eter. The DNA content in the supernatant was determined by thediphenylamine method of Burton (9). Specific activity of DNA was expressed as cpm/ng of DNA.

LI. Each animal received 30 /iCi of [mef/7y/-3H]thymidine 30 min priorto killing, as described above. Dorsal skin pieces were fixed in Bouin's

solution (2 hr) and then in 4% formaldehyde, embedded in paraffin, cutat 3 /im, stained with hematoxylin, dip-coated with Kodak NTB-2 emul

sion diluted 1/1 (v/v) with distilled water, and developed after 2 weeksof exposure. The LI was scored as the number of labeled basal cells per1000, using 3 grains over the nucleus as minimum.

Assay of ODC Activity. Epidermises isolated from 2 mice as describedabove were pooled. Following homogenization and centrifugation, thesoluble epidermal extracts were assayed for ODC activity, which wasdetermined by measuring the release of 14CU2from OL-[1-14C]omithine

as described (4). Enzyme specific activities were expressed as nmol ofCO2 evolved in 30 min/mg of soluble protein.

Protein Determination. The protein concentrations of the solubleepidermal extracts were measured by the method of Lowry ef al. (20)with bovine serum albumin as standard.

RESULTS

Effects of Different Doses of MEZ

In order to find the maximal, nonulcerating dose of MEZ, singleapplications of 1.7,8.5, or 17 nmol were tested by observing thetreated epidermis the following week. MEZ at 17 nmol inducedconsiderable ulcérationafter 48 hr. No ulcérationwas observedafter a single treatment of 1.7 or 8.5 nmol.

Polyamine Levels. Chart ~\Ashows the changes in epidermal

putrescine levels. A considerable increase with a distinct peakat 16 hr and a shoulder at 64 hr was seen after treatment with17 nmol. MEZ (8.5 nmol) induced a distinct biphasic increase,with peaks at 16 and 32 hr. After 1.7 nmol, there seemed to benegligible changes. The degree of accumulation of putrescinecorrelated well with increasing doses of MEZ. Chart ÃŒBshowsthe changes in epidermal spermidine levels. The 3 doses of MEZ

1600-

1200-

800

400

100

g«200"o

g 150o

5 100

120

100

BO

60

40

20

O 8 9616 24 32 48 64 72Hours after treatment

Chart 1. Changes in epidermal levels of putrescine (A), spermidine (8), andspermine (C) following single applications of either 1.7 (•),8.5 (•),or 17 nmol (A)of MEZ. Eight mice (skin of 2 mice pooled for each analysis; n = 4) were treatedwith either 0.2 ml of acetone (control) or MEZ dissolved in 0.2 ml of acetone, killedat the times indicated, and assayed as described in 'Materials and Methods." The

changes in MEZ-treated epidermis are expressed as a percentage of the controlsat each time point. The mean of 32 controls was 4.23 ±2.3 (S.D.) nmol/mg ofDNA for putrescine, 124 ±33 nmol/mg of DNA for spermidine, and 72 ±20 nmol/mg of DNA for spermine. Bars, S.D.

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J. E. Paulsen and E. G. Astrup

induced fluctuations and a small increase of the spermidinelevels. No clear systematic relationship between alterations inspermidine levels and doses of MEZ was seen. Chart 1C showschanges in the spermine levels. Tretment with 8.5 and 17 nmolof MEZ led to a pronounced decrease in the spermine levels,which normalized at 64 and 96 hr, respectively. MEZ (1.7 nmol)induced small fluctuations around control level. A good correlation between the duration of decreased spermine levels andincreased doses of MEZ was observed. Chart 2A shows changesof the molar ratio of spermidine to spermine. MEZ (1.7 nmol)induced a single-peaked increase, whereas 8.5 and 17 nmol

caused pronounced biphasic increases. The peak heights correlated well with the increasing doses. A systematic delay of thepeaks with increasing doses was observed. The pronouncedenhancement of the molar ratio of spermidine to spermine wasa result of decreased spermine levels as well as of increasedspermidine levels.

DNA Synthesis (Specific Activity). The results are shown inChart 2ß.A good correlation was observed between increasingdoses of MEZ and the degree and duration of the initial inhibitionof DNA synthesis. A first peak was seen at 16 hr with the 1.7-and 8.5-nmol doses and at 24 hr with the 17-nmol dose. MEZ

(1.7 nmol) induced the highest first peak. The height and durationof the second peak correlated well with increasing doses. Prob-

450-

400

350-

300

250

200

150

100

« 350oio- 300

250

200

150

100

50

OO 8 16 24 32 48 64 72 96

Hours alter treatment

Chart 2. Changes in epidermal molar ratio of spermidine/spermme (A) andepidermal DNA synthesis (8) following single applications of either 1.7 (•).8.5 (•),or 17 nmol (A) of MEZ. Treatments and groups of mice as in Chart 1. The meanof 32 controls was 1.69 ±0.24 (S.D.) for spermidine/spermineand 50.2 ±11 cpm/ug of DNA for specific activity of DNA. Bars, S.D.

750-

600-

0 8 16 24 32 48 64 72

Hours after treatmentCharts. Changes in epidermal levels of putrescine (A), spermidine (B), and

spermine (C) following single applications of either 17 nmol of TPA (•),8.5 nmoteof MEZ (A), or 20 «¿molof EPP (•).Eight mice (skin of 2 mice pooled for eachanalysis;n = 4) were treated with either 0.2 ml of acetone (controls)or TPA, MEZ,or EPP dissolved in 0.2 ml of acetone, killed at the times indicated, and assayedas described in "Materials and Methods." The changes in TPA-, MEZ-, or EPP-

treated epidermis are expressed as a percentage of controls at each time point.The mean of 32 controls was 4.23 ±2.3 (S.D.) nmol/mg of DNA for putrescine,124 ±33 nmol/mg of DNA for spermidine, and 72 ±20 nmol/mg of DNA forspermine.Bars, S.D.

ably due to the initial inhibiting effect, the height of the first peakdid not correlate with increasing doses. The spermidine/spermineratio and DNA synthesis followed parallel time courses for all 3of the doses tested, although the peak heights of these 2parameters did not correlate in the initial period.

Effects of Single Applications of TPA, MEZ, and EPP

For comparison, the results of 8.5-nmol MEZ treatment are

drawn together with the results of TPA and EPP treatment inCharts 3 and 4. A tumor-promoting dose of TPA (17 nmol) and

a maximal, nonulcerating dose of EPP (20 ^mol) were chosenaccording to previous reports (16, 26, 28).

Polyamine Levels. The results are shown in Chart 3. TPA andMEZ induced a first peak of putrescine levels at 8 to 16 hr anda second peak at 32 to 48 hr; MEZ caused the strongest effect.EPP caused a slight increase at 8 to 16 hr. Changes in spermidine

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Epidermal DNA and Polyamlne Synthesis after TPA, MEZ, or EPP

0 S 16 24 32 48 64 72

Hours after treatment

Chart 4. Changes in epidermal molar ratio of spermidine/spermine (A) andepidermal DMAsynthesis (B) following single applicationsof either 17 nrnol of TPA(•),8.5 nmol of MEZ (A), or 20 /imol of EPP (•).Treatments and groups of miceas explained in Chart 3. The mean of 32 controls was 1.69 ±0.24 (S.D.) forspermidine/spermineand 50.5 ±11 cpm/pg of DNA for specific activity of DNABars, S.D.

levels (Chart 38) after TPA and EPP treatment showed similartime courses with a single peak; TPA caused the most pronounced increase. MEZ caused a fluctuating pattern. A systematic pattern was observed in the spermine levels (Chart 3C) afterthe 3 treatments; an initial decrease was followed by a slightstimulation. TPA and EPP led to changes with similar timecourses, but both the first decrease as well as the first increasewere more pronounced after treatment with TPA. MEZ causedthe most pronounced initial decrease in both degree and duration. TPA and EPP induced pronounced biphasic increases inthe molar ratio of spermidine to spermine, whereas EPP induceda single-peaked increase (Chart 4A).

ODC Activity. Table 1 shows that TPA (17 nmol), MEZ (17nmol), and EPP (20 /¿mol)induce ODC to the same degree at4.5 hr. Since we do not know the time course with maximumODC induction after MEZ and EPP treatment as we know forTPA (4), this study only indicates the quantitative aspects.

DNA Synthesis (Specific Activity). The results are shown inChart 48. TPA, MEZ, and EPP caused similar initial inhibitionsof DNA synthesis at 8 hr. TPA and EPP induced an identical firstpeak at 16 hr, whereas the first peak (16 hr) after MEZ was lesspronounced. TPA induced a high second peak at 32 hr. MEZ

induced a wide second peak at 32 to 48 hr (peak height wasless than that after TPA). As already demonstrated for variousdoses of MEZ (Chart 2), TPA and EPP led to parallel time coursesof spermidine/spermine ratio and DNA synthesis (Chart 4). In theinitial period with inhibited DNA synthesis, there was no correlation between the 2 variables, but a good correlation was seenlater on. When the effects of TPA, MEZ, and EPP were compared, there was no correlation between peak heights of DNAsynthesis and peak heights of spermidine/spermine ratio, incontrast to what was found in the dose-effect study of MEZ.

LI. The LI results (Chart 5) show that the waves of DNAsynthesis (specific activity) seen after TPA, MEZ, and EPPtreatment (Charts 28 and 48) are associated with an increasednumber of labeled cells. After 17 nmol of TPA, as well as after17 nmol of MEZ and 20 ftmol of EPP, the LI and specific activityof DNA had almost identical time courses, showing that thenumber of cells in S phase was increased at the peak values.

DISCUSSION

This work supports the hypothesis that the polyamines maybe associated with the proliferate changes after epidermalgrowth stimulation. By measuring the specific activity of DNAand polyamine levels in the same cells and comparing the effectsof TPA, MEZ, and EPP in the same experiment (thus avoidingsome biological and methodological variations), this work canprovide new and more detailed information about the role of

Table 1EpidermalODCactivity following single skin applications of TPA,MEZ, or EPPThe animals were killed 4.5 hr after treatment, and the ODC activity was

determined as described in 'Materials and Methods."

Treatment

ODC activity (nmol ofCO..in 30 min/mg of

protein)

TPA (17 nmol)MEZ (17 nmol)MEZ (8.5 nmd)EPP(20 ^mol) in acetone (0.2 ml)Acetone

2.56 ±1.02*

2.11 ±0.321.78 ±0.641.10 ±0.110.10 ±0.03

a Mean ±S.D. of 4 groups of mice, using 2 mice in each group.

oo° 700-

<5o.—» 500-o

o>« 300-1

E3

Z

100-

0 8 16 24 32 48 72Hours after treatment

96

Chart 5. Changes in U of basalcells, determinedas described in •MaterialsandMethods,* after single applications of either 0.2 ml of acetone (controls) (*) or 17

nmol of TPA (•),17 nmol of MEZ (•),or 20 iimot of EPP(A) dissolved in 0.2 ml ofacetone. The curves are drawn through the mean values of the determinationsmade from 2 mice.

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J. E. Paulsen and E. G. Astrup

polyamines in cell proliferation and tumor promotion. The tumorpromoter TPA induces no characteristic changes in polyaminesynthesis and DNA synthesis compared to the changes aftertreatments with the mitogens MEZ and EPP. Both TPA and MEZinduce pronounced biphasic increases in DNA synthesis, accumulation of putrescine, and the spermidine/spermine ratio,whereas EPP induces single-peaked increases in the same var

iable. All treatments tested here caused the following sequentialchanges: initial inhibition of DNA synthesis; induction of ODCactivity; and subsequent peaks of putrescine levels precedingpeaks of DNA synthesis. This is in accordance with our previousresults after TPA treatment (4); however, there are some discrepancies between the time courses of the present and priorTPA curves, probably caused by the reduced number of investigated time points in the present study and a certain variation incell kinetics. Neither the steady increase of spermidine levels northe steady decrease of spermine levels correlated with the wavesof DNA synthesis. However, a remarkably good parallelism between the time courses of the molar ratio of spermidine tospermine and the DNA synthesis was seen, except during theinitial period, when the DNA synthesis was inhibited. Thus, afterall 3 of the treatments, there seemed to be a positive correlationbetween putrescine levels and spermidine/spermine ratios onthe one hand and DNA synthesis on the other.

The good correlation between the dose of MEZ and the degreeand duration of the initial inhibition of DNA synthesis, togetherwith the observation that the highest dose of MEZ causesulcérationof the skin, suggests that the initial inhibition of DNAsynthesis is a sign of cellular toxicity. Because TPA and EPPinduce similar initial inhibition of DNA synthesis, we believe thatthis is a general and nonspecific effect of skin irritants. This viewis supported by the results of several other experiments usingTPA (2, 3, 28), EPP (27), cantharidine (11), tape stripping (10),and sandpaper rubbing (6). It is reported that TPA (28), EPP (27),and cantharidine (15) cause a dose-dependent initial inhibition of

DNA synthesis similar to the one we find for MEZ.As reported previously (2), TPA causes an initial block of cells

in S and G2 phase as well as of mitotic activity, followed by apartly synchronized cohort of cells going through multiple wavesof DNA synthesis and mitosis. The LI and specific activity ofDNA presented here indicate changes in cell kinetics after MEZand EPP similar to those seen after TPA. From a cell kineticpoint of view and with background in previous studies (2), wecan put forward the following hypothesis for the observedTPA-, MEZ-, and EPP-induced responses in epidermis: (a) after

an initial block in DNA synthesis and mitotic activity, the basalcells are partly synchronized; (b) the observed subsequentchanges in DNA and polyamine biosynthesis are the manifestations of a partly synchronized, large cohort of basal cells passingthrough multiple cell cycles; (c) the observed peaks of putrescinefollowed by peaks of DNA synthesis might indicate that anincrease in putrescine levels takes place in late G,; and (d) thehigh correlation between the variations in spermidine/spermineratio and in DNA synthesis (specific activity and LI) indicates thata proper balance between these 2 compounds must exist whencells are passing S phase. Since extracts from the whole epidermis were used, it is not possible to say whether the observedchanges in polyamine synthesis are solely due to alterations inthe rate of cell proliferation in the basal layer, to alterations inthe rate of cell differentiation (3), or to a combination of these

possibilities. Generally, polyamines can be associated both withcell proliferation (13,18, 30, 35) and cell differentiation (14).

In the dose-effect study of MEZ, we find parallel time courses

for putrescine accumulation, spermidine/spermine ratio, andDNA synthesis, with the putrescine peaks somewhat shifted tothe left. A high correlation between the peak heights for the 3parameters is evident. When the effects of TPA, MEZ, and EPPare compared, we still find similar parallel time courses, but nocorrelation exists between peak heights of putrescine and thespermidine/spermine ratio on the one hand and peak heights ofDNA synthesis on the other. There may be at least 2 alternativeexplanations for this phenomenon: (a) the spermidine/spermineratio and the level of putrescine may play different roles in theprocess of proliferation after the treatments with TPA, MEZ, orEPP; or (b) the spermidine/spermine ratio and the level of putrescine may be more closely related to other biological processes which change synchronously with the waves of DNAsynthesis as, for example, the rate of cell differentiation (3). Tosolve these problems, we need more knowledge about thedistribution of polyamines in the epidermis after stimulated proliferation. Alternatively, the causal relationship between polyamine synthesis and DNA synthesis could be studied by usinginhibitors of polyamine metabolism. Some studies with retinoleacid (5, 8), DL-hydrazinoaminovaleric acid (35), and a-difluoro-

methylomithine (30) may suggest a causal relationship betweenaccumulation of putrescine and DNA synthesis in mouse epidermis.

Comparing the effects of TPA, MEZ, and EPP, induction ofODC and polyamines does not seem to be specific for promotionbut merely seems to be associated with the cell kinetic eventsduring stimulated cell proliferation. Since all tumor-promoting

agents studied on epidermis have strong mitogenic effects,promotion seems to be invariably linked to epithelial hyperpro-

liferation (21). Because of the good correlation between polyamine synthesis and DNA synthesis, we believe that a discussionof the role of polyamines in tumor promotion will also be adiscussion of the role of hyperproliferation. Skin tumor promotionmay depend in part on a certain number of cell turnovers (hyper-

proliferation), thereby increasing the statistic probabilities ofpropagating precancerous cells. The quantitative changes in cellproliferation and polyamine synthesis may be essential for theso-called second stage of promotion, described by Slaga et al.

(33, 34) and Weeks ef al. (39). The fact that MEZ is a goodsecond stage promoter, while EPP is not, suggests that thesimilar biphasic patterns of both DNA synthesis and polyaminesynthesis observed after TPA and MEZ, in contrast to the single-

peaked patterns observed after EPP, may be related to thesecond stage of promotion. However, in order to evaluate thesignificance of hyperproliferation and polyamine synthesis fortumor promotion, more studies of cell population kinetics andpolyamine analysis after multiple applications of various promoters and mitogens are needed in the future.

ACKNOWLEDGMENTS

We are indebted to the technical staff at the Institute of Pathology for excellentassistance, to the secretariat for typing and retyping of manuscripts, to theDepartment of Laboratory Animals and the staff there for taking care of the animals,and to the University Phototechnical Department, Rikshospitatet, and the PathologyExtension of the University Library for their help. We want to thank Dr. Knut Eliasenand Dineke Gronnseth at the Veterinary College of Norway for letting us use theHPLC equipment and for their invaluable assistance with the HPLC analysis.

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12. Clausen, O. P. F., Thorud, E., Bjerknes, R., and Elgjo, K. Orcadian rhythms inmouse epidermal basal cell proliferation. Cell Tissue Kinet., 12:319-337,1979.

13. Gaugas, J. M. Polyamines in Biomedicai Research. New York: John Wiley andSons, 1980.

14. Heby, O. Roles of polyamines in the control of proliferation and differentiation.Differentiation, 79:1-20,1981.

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SEPTEMBER 1983 4131

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1983;43:4126-4131. Cancer Res   Jan Erik Paulsen and Erle Grieg Astrup  in Hairless Mouse EpidermisEthylphenylpropiolate on DNA Synthesis and Polyamine Levels -Tetradecanoylphorbol-13-acetate, Mezerein, or

OEffects of Single Applications of 12-

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