effects of the phorbol ester 4-o-methyl-12-o ... · , r* Â¿*

[CANCER RESEARCH 42, 342-348, January 1982]0008-5472/82/0042-OOOOS02.00

Effects of the Phorbol Ester 4-O-Methyl-12-O-Tetradecanoylphorbol-13-

acetate on Mouse Skin in Vivo: Evidence for Its Uselessness as a

Negative Control Compound in Studies on the Biological Effectsof Phorbol Ester Tumor Promoters1

Gerhard FÃ¼rstenberger,2Hartmut Richter, Thomas S. Argyris, and Friedrich Marks

Deutsches Krebsforschungszentrum (German Cancer Research Center), Institut fÃ¼rBiochemie, Im Neuenheimer Feld 280, D-6900 Heidelberg, Germany [G. F., H.R., F. M.], and Department Ã¶lPathology, State University of New York, College of Medicine, Syracuse, New York 13210 [T. S. A.]

ABSTRACT

Compared to 12-O-tetradecanoylphorbol-13-acetate (TRA),the phorbol ester 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate (4-O-methyl-TPA) is only a weak skin irritant and tumorpromoter. When topically applied to mouse skin, 400 nmol 4-O-methyl-TPA induce hyperproliferation of epidermis in vivo to

approximately the same extent as do 10 nmol TPA. However,in contrast to TPA, 4-O-methyl-TPA-induced proliferation is not

followed by sustained epidermal hyperplasia. In addition, themethyl ether of TPA does not cause early prostaglandin accumulation, and its hyperproliferative effect is insensitive to in-domethacin inhibition.

Contrary to the action of TPA, 4-O-methyl-TPA induces nei

ther an early increase in the rate of phospholipid turnover noran early increase in ornithine decarboxylase activity or polya-mine accumulation in epidermis and does not desensitize epidermis to the effects of /J-adrenergic agonists and epidermalGÃ¬chalone. Finally, 4-O-methyl-TPA is only a weak stimulatorof epidermal cyclic adenosine 3':5'-monophosphate phospho-

diesterase activity. Consequently, it is concluded that themechanism of action of 4-O-methyl-TPA as a skin mitogen isdifferent from that of TPA. Whereas TPA evokes prostaglandin-dependent and chalone-insensitive hyperplastic development

of epidermis (hyperplastic transformation), i.e., probably a relapse to the neonatal state, 4-O-methyl-TPA apparently stimu

lates nothing but normal everyday tissue regeneration. In thisrespect, treatment with 4-O-methyl-TPA resembles a non-

damaging stimulus of epidermal cell proliferation (observed, forexample, with skin massage), whereas TPA evokes a responsewhich is characteristic for epidermal injury.

Considering these fundamental differences in biological action between TPA and its methyl ether, 4-O-methyl-TPA is

concluded to be an unsuitable negative control compound forstudies on the biological effects of phorbol ester tumor promoters.

INTRODUCTION

The investigation of the mechanism of action of phorbol estertumor promoters has recently become a major issue of experimental cancer research. It has been found that these compounds exhibit a wide variety of "hormone-like" effects on

different tissues and in vitro systems (3, 7, 15, 36). However,

1This work was partially supported by grants from the Deutsche Forschungsgemeinschaft and the Wilhelm- and Maria-Meyenburg-Foundation.

2 To whom requests for reprints should be addressed.Received June 15, 1981; accepted September 6, 1981.

the causal relationship of most of these effects with the phenomenon of tumor promotion has not yet been convincinglyestablished. There are 2 reasons for this discrepancy, (a) Withonly a few exceptions, most biological systems studied thus farare, at least at the moment, not suitable for accomplishing aclearcut 2-stage carcinogenesis experiment, (b) In mouse skin,the classical target tissue of tumor promotion, all tumor promoters have been found to be irritant mitogens, whereas notevery irritant skin mitogen is a tumor promoter (24). Thisindicates that hyperplasiogenic activity is probably a necessarybut certainly not a sufficient property of a tumor promoter. Thediscovery of the 2-stage approach of skin tumor promotion (4,11, 35) has now unequivocally shown that the promotion-

specific effects of a phorbol ester tumor promoter such asTPA3 are different from its pleiotropic effects on cell prolifera

tion. The mitogenic and irritant activities even apparently camouflage the promotion-specific actions. This situation makes it

very difficult to decide whether any effect of a phorbol esterobserved in a given biological system is related to promotionor merely reflects the pleiotropic efficiency of the compoundas a skin mitogen. Even a positive correlation of an observedeffect with tumor-promoting efficacy cannot be taken as a

convincing proof that this effect is indicative for promotion,since skin mitogenic activity and promoting potency more orless run parallel within the series of phorbol esters.

Since there has been a rapid increase of data on the biological effects of phorbol esters (for reviews, see Refs. 3, 7, 15,and 36), the problem of identifying promotion-specific eventsand of distinguishing them clearly from those related to pleio-tropism has become of outstanding importance for the interpretation of experimental results, the design of theoreticalconcepts, and the question as to whether a study may providea better insight into a major problem of experimental cancerresearch or nothing but new data on basic problems of cellbiology.

The use of an appropriate negative control compound couldbe of help in escaping this dilemma. Such a control compoundmust be a nonpromoting irritant skin mitogen, which shouldinduce epidermal hyperproliferation essentially along the samepathways as a promoter. Nonmitogenic compounds such asphorbol or highly toxic irritants such as acetic acid [which killthe initiated cells before they can be promoted (31)] are, ofcourse, quite useless as controls.

3 The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; 4-O-methyl-TPA, 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate; RPA, 12-O-retinoylphorbol-13-acetate; cyclic AMP, cyclic adenosine 3':5'-monophosphate.

342 CANCER RESEARCH VOL. 42

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http://cancerres.aacrjournals.org/

4-O-Methyl-TPA Effects on Mouse Skin

Recently, the 4-O-methyl ether of TRA, a weakly promotingskin mitogen (25), as well as the closely related compound 4-O-methylphorbol-12,13-didecanoate have been proposed to

be the most appropriate negative control compounds (14) andhave been used as such in many laboratories (see, e.g., Refs.1, 5, 6, 8, 9, 13, 18, 19, 21, 29, 33, 34, 38, 39, and 41 ). Herewe show that the proliferative response of mouse epidermis to4-O-methyl-TPA is fundamentally different from that to TPA.

Therefore, we suggest replacing this phorbol ester by a moresuitable negative control compound, i.e., a nonpromoting hy-

perplasiogenic skin irritant, such as the retinole acid derivativeof TPA (11, 24).

MATERIALS AND METHODS

Chemicals. The phorbol esters TPA and 4-O-methyl-TPA were gen

erously supplied by Professor E. Hecker, German Cancer ResearchCenter, Heidelberg (Federal Republic of Germany). The synthesis andpurification (by preparative high-pressure liquid chromatography) of

50CH

â€¢â€¢2nmolâ€”Â« lOnmolâ€”o-- 20nmo(

! 340

Days

Chart 1. Effect of TPA [2 to 20 nmol (left)] and 4-O-methyl-TPA (4-O-Me-TPA)[400 nmol (right)] on DMA labeling in dorsal mouse epidermis in vivo. The phorbolesters were applied in 0 1 ml acetone solution each at 0 time, and the animalswere killed at the times indicated. One hr prior to being killed, 30 Â»Ci| 'H|-

thymidine were injected i.p. into each of 5 animals, ta, average S.E. of thecontrols (treated with acetone instead of with phorbol ester) with 51 Â±13 cpm/pg DNA A 100% (n = 120). Bars, S.E. Inset, dose-response curve for 4-O-methyl-TPA as measured 18 hr after treatment (n = 10).

O i 2 Ã 4 5Days

B i/

^\.

lLÂ¿Â¿3*100-50-nC1t/lI,L-Jt'-i-ri

01234Days nmol

Chart 2. Induction of epidermal hyperplasia by TPA and 4-O-methyl-TPA inthe dorsal interfollicular epidermis of female NMRI mice. A and 8, effect of asingle topical application of either TPA (â€¢ â€¢10 nmol/0.1 ml acetone), or 4-O-methyl-TPA (â€¢ â€¢.400 nmol/0.1 ml acetone), or acetone (O O, 0.1

ml) at 0 time on the number of nucleated cells per mm interfollicular epidermis(A) or the number of nucleated cell layers (8) as measured at the times indicated.C, number of nucleated cells in interfollicular epidermis counted per visual field(X 1000) 48 hr after phorbol ester application as a function of phorbol esterdose. , 4-O-methyl-TPA; , TPA. Number of visual fields counted = 35.Bars, S.E.

f? â€¢'C;**;-', r* Â¿*<*v/*Â»- â€¢

<*â€¢ o â€¢̂ ^ . â€¢tÂ». * f^f

Fig. 1. Morphological appearance of mouse skin 48 hr after a single topicalapplication of 0.1 ml acetone [a], 4-O-methyl-TPA [fa(400 nmol)], and TPA [c (10

nmol)].

RPA will be described in a forthcoming paper.4

A fraction enriched in epidermal GÃ¬chalone was obtained by ethanolprecipitation (55%) of an aqueous extract from defatted pig skin (16,22). When injected i.p. into adult NMRI mice, 0.1 to 0.2 mg of thismaterial caused a 50 to 60% inhibition of epidermal DNA labeling invivo within 16 hr (for details, see Ref. 22). For all other chemicalsincluding radioactively labeled compounds, see Ref. 27.

Animals. Female NMRI mice (7 to 8 weeks old) whose back skinwas shaved 3 to 5 days prior to the experiments were used. Other

' B. Sorg, G. FÃ¼rstenberger, D. L. Berry. E. Hecker, and F. Marks. Preparation

of retinoic acid esters of phorbol derivatives, submitted for publication.

JANUARY 1982 343



G. FÃ¼rstenberger et al.

Table 1Effect of topical application of TPA (10 nmol/0.1 ml acetone) or 4-O-methyl-TPA (400 nmol/0.1 ml acetone) on the

level of prostaglandin CÂ¡(IO min after treatment), DNA labeling (18 to 21 hr after treatment), and mitotic activity (24to 27 hr after treatment) in dorsal mouse epidermis in vivo and influence of indomethacin (1.1 Â¡unol/0.1 ml acetone

topically applied 30 min prior to phorbol ester treatment) or prostaglandin E2 (30 nmol/0.1 ml acetone topicallyapplied simultaneously with the phorbol ester) on these parameters

For details, see "Materials and Methods."

TreatmentAcetone

(control)Acetone + indomethacinTPATPA + indomethacinTPA + prostaglandin E24-O-Methyl-TPA4-O-Methyl-TPA + indomethacin4-O-Methyl-TPA + prostaglandin E2Prostaglandin

E.(pg/iigDMA)7.3

Â± 2.0a (34)"

6.9 Â± 1.1 (6)52.6 Â±21.6 (24)

9.2 Â± 2.7 (12)NDC

8.7 Â± 3.6 (12)NDNDDNA

labeling(cpm/figDNA)51

Â±13(120)50 Â±16 (10)

242 Â±60 (1 0)49 Â±16 (10)

380 Â±55 (1 0)155 Â±48 (10)144 Â±28 (10)148 Â±58 (10)Mitotic

activity(metaphase figures/

visualfield)0.21

Â±0.06(16)0.22 Â±0.05(16)1.81 Â±0.49(16)0.16 Â±0.05(16)

ND1.24 Â±0.36(16)1.34 Â±0.42(16)

ND

Mean Â±S.E.6 Numbers in parentheses, number of Experiments (animals).c ND, not determined.

details of handling and treatment have been described elsewhere (20).Determination of Epidermal Phosphatidylcholine. For phospholipid

analysis, the dissected skin was immediately frozen at -80Â° on a cold

table (27). Then the epidermis was scraped off and homogenized in 2ml ice-cold 0.4 M HCIO4. The homogenates were allowed to stand in

ice for 30 min and then centrifuged, and the pellet was washed 3 timeswith 2 ml cold 0.4 M HCIO4. The lipids were thoroughly extracted 3times with 2 ml chloroform/methanol (2/1, v/v), each at room temperature. DNA was assayed in the remaining pellet (20). The lipid extractwas washed according to the method of Folch ef al. (10). The organicsolvent was removed by evaporation, and the residue was redissolvedin 0.1 ml chloroform/methanol (1 /1, v/v) and subjected to Chromatographie separation on silica gel using Merck ready-made thin-layer

plates, which were developed in preequilibrated chambers containingchloroform/methanol/acetic acid/water (75/45/12/3, v/v). Thebands were visualized by iodine vapor, and the phosphatidylcholineband was identified by comparison with authentic phosphatidylcholinerun on the same plate. The phosphatidylcholine band was scraped off,and the scrapings were used for determination of radioactivity.

Other Methods. The methods for labeling epidermal DNA /;; vivo(20), determination of mitotic activity (2, 20), assay of ornithine decar-

boxylase (25) and cyclic AMP phosphodiesterase activities (26), andassay of prostaglandin E2 (27) and cyclic AMP (23) have been described in detail elsewhere.

RESULTS

Epidermal Hyperproliferation. The methylation of the hy-droxyl group in position 4 of the TPA molecule results in analmost complete loss of the irritant activity and a considerableweakening of the mitogenic potency of the phorbol ester. Asshown in Chart 1, 4-O-methyl-TPA had to be used in a dose ofat least 40 times that of TPA in order to induce nearly the sameproliferative response in the dorsal epidermis of NMRI mice. Incontrast to TPA, the methyl ether did not cause an initialdepression of DNA labeling.

One of the most striking differences observed was that, whileTPA-induced epidermal hyperproliferation was followed by sus

tained epidermal hyperplasia, i.e., a considerable increase ofthe number of cells and a doubling of the number of cell layersin interfollicular epidermis, 4-O-methyl-TPA evoked almost no

hyperplastic response even when applied in doses up to 800nmol/animal (Chart 2; Fig. 1).

4-O-Methyl-TPA-induced Epidermal Hyperproliferation Isnot Prostaglandin-mediated. TPA-induced hyperproliferation

of mouse epidermis in vivo has been shown to be dependenton a transient accumulation of prostaglandin E2 immediatelyafter treatment (24, 27). When prostaglandin synthesis wasprevented by pretreatment of skin with the cyclooxygenaseinhibitor indomethacin, no increase in the rate of cellular proliferation and no hyperplasia could be observed. In contrast, 4-O-methyl-TPA was unable to stimulate epidermal prostaglandin

synthesis in vivo (Table 1) or the release of arachidonic acidfrom prelabeled phospholipids in primary epidermal cell cultures.5 Consequently, epidermal hyperproliferation induced by

the 4-O-methyl ether of TPA was found to be insensitive to

indomethacin inhibition (Table 1). This result was confirmed byexperiments in which the phorbol ester and prostaglandin E2were applied simultaneously. Whereas the stimulatory effect of4-O-methyl-TPA on DNA labeling remained unchanged, prostaglandin E2 augmented the TPA-induced DNA synthesis (Table

1).Other Effects Possibly Related to Epidermal Hyperpro

liferation. TPA and other skin mitogens induce in mouse epidermis ornithine decarboxylase activity within 2 to 6 hr aftertreatment (25, 32). This effect has been proposed to be relatedto epidermal cell proliferation and skin tumor promotion (32).No increase of enzyme activity and no accumulation of polya-mines were seen after in vivo application of 4-O-methyl-TPA

(25).Another effect possibly linked to the induction of epidermal

cell proliferation is an early increase of the rate of phospholipidturnover in vivo. Such an effect could already be clearly observed 1 to 2 hr after TPA treatment, whereas 4-O-methyl-TPA

was unable to provoke such an early response. Thus, after atleast 3 to 4 hr, the turnover of phosphatidylcholine in 4-O-methyl-TPA-treated epidermis was found to be only slightly

increased (Table 2).4-O-Methyl-TPA Does Not Induce G, Chalone Refractori

ness. Hyperplasiogenic agents such as TPA have been shownto desensitize mouse epidermis to the inhibitory effect of epidermal GÃ¬chalone, a tissue-specific endogenous inhibitor of

the G, S transition (2, 20). This effect has been proposed to becausally related to the development of the hyperplastic state.Epidermis retained its chalone sensitivity when DNA labelingwas measured 18 hr after application of different doses of 4-

5 C. Delescluse, G. FÃ¼rstenberger. and F. Marks, unpublished results.





O-methyl-TPA (Chart 3) or between 16 and 24 hr after 400nmol 4-O-methyl-TPA (data not shown).

4-O-Methyl-TPA Does Not Exhibit Pronounced Effects onthe Epidermal Cyclic AMP System. TPA has some effects onthe cyclic AMP system in epidermis in vivo. Thus, it leads to alimited accumulation of cyclic AMP after 1 to 2 hr (13), to aconsiderable stimulation of cyclic AMP phosphodiesterase activity (26, 37) and to a pronounced and long-lasting desensiti-zation of cyclic AMP formation to ÃŸ-adrenergic stimulation (13,30). In contrast, 4-O-methyl-TPA has almost no influence onthe yÃŸ-adrenergiceffect (Chart 4) and induces only a rathermoderate and short-lived increase of phosphodiesterase activ

ity (Chart 5).Tumor-promoting Efficacy of 4-O-Methyl-TPA. We have

shown recently that a 2-stage carcinogenesis experiment performed with 4-O-methyl-TPA as a promoter (400 nmol/appli-

Table 2Effect of a single-topical application of acetone (0.1 ml), TPA (10 nmol/0.1 ml

acetone), or 4-O-methyl-TPA (400 nmol/0.1 ml acetone) at 0 time on the

labeling of phosphatidylcholine in mouse epidermis in vivo

The animals were killed at the times indicated. One hr prior to killing, eachanimal received an i.p. injection of 120 /iCi [3H]choline.

Labeling of phosphatidylcholine (cpm//ig DMA) after treatmentwith(hr)

Acetone1

16.82 16.93 15.34 17.1Â±

Â±Â±Â±6.3a

4.65.72.0TPA23.9

48.643.838.7Â±

Â±Â±Â±5.9

12.813.5

6.14-O-Methyl-TPA18.1

18.424.928.2Â±

Â±Â±Â±4.9

5.45.64.0

" Mean Â±S.E. of 6 mice.

0-

o>efJO

oo

JC

1000 1 1O 100 1OÃ–O

nmol

Chart 3. Effect of semipurified epidermal G, chalone on DNA labeling in TPA( ) and 4-O-methyl-TPA-treated ( ) mouse epidermis in vivo. The animalswere killed 18 hr after TPA or 21 hr after 4-O-methyl-TPA application. Sixteen hrprior to being killed the animals received an i.p. injection of 0.5 mg chalonepreparation (see "Materials and Methods") in 0.2 ml 0.9% NaCI solution con

taining 0.05% sodium dodecyl sulfate. One hr before sacrifice, 30 /iCi[3H]thymidine were injected i.p. (see Chart 1). Control animals were also treated

with phorbol ester but received only an injection of 0.9% NaCI solution insteadof chalone. Ordinate, inhibition of DNA labeling by chalone treatment as compared with the controls (= 0%). Abscissa, phorbol ester dose applied, ea,averageS.E. of the controls (n = 21 ). Points, mean values of 5 to 9 experiments (animals),ears, S.E.

l T \t.

Chart 4. Effect of an i.p. injection of DL-isoproterenol on the accumulation ofcyclic AMP in acetone (O O), TPA (â€¢ â€¢).or 4-O-methyl-TPA-treated( ) mouse epidermis in vivo. Isoproterenol (1 /imol/0.25 ml 0.9% NaCIsolution) was injected at 0 time, and the animals were killed at the times indicated.Phorbol esters (20 nmol TPA; 400 nmol 4-O-methyl-TPA) were tooically applied6 hr before isoproterenol injection. The 4-O-methyl-TPA curve shows individual

points, whereas the other points are mean values of at least 6 experiments(animals). Bars, S.D. ea, cyclic AMP level in the epidermis of control animals (nophorbol ester treatment and no isoproterenol, n = 20). Inset, cyclic AMPaccumulation 7 min after isoproterenol injection in TPA ( ) and 4-O-methyl-TPA ( Mreated animals as a function of the time interval between phorbolester application and isoproterenol injection (abscissa). To diminish a possibleeffect of cyclic AMP phosphodiesterase on cyclic AMP accumulation, theseexperiments were carried out with animals which had been pretreated with 1-methyl-3-isobutylxanthine (1.5 mg topically applied in 0.1 ml acetone 40 min and1 mg/0.3 ml 0.9% NaCI solution injected i.p. 15 min prior to isoproterenoladministration). Number of animals, ^5. Lower la, average S.D. of the cyclic AMPlevel in control animals, which had received only 1-methyl-3-isobutylxanthine butno isoproterenol, and received acetone instead of phorbol ester (n z 20). Upperea, average S.D. of the cyclic AMP level 7 min after i.p. injection of isoproterenolinto 1-methyl-3-isobutylxanthine-treated animals which received acetone insteadof phorbol esters (n 5 20).

cation) yielded only a very low number of papillomas (25). Arepetition of this experiment is shown in Table 3B.

In addition, the efficacy of 4-O-methyl-TPA as either a first-or a second-stage (incomplete) tumor promoter was tested,

since it has been shown that tumor promotion can be subdivided into 2 stages (4, 11, 35). As shown in Table 3A, 4-O-methyl-TPA is unable to promote the second stage in anexperiment with TPA as first-stage promoter. When a limitednumber of applications of 4-O-methyl-TPA was followed byprolonged treatment with RPA, the most potent incompletetumor promoter known (24), weak efficacy of 4-O-methyl-TPAas first-stage promoter was revealed (Table 3B) which was,

however, not comparable with that of TPA (Table 3C).

DISCUSSION

In an appropriate dose, the almost nonpromoting phorbolester 4-O-methyl-TPA induces hyperproliferation of mouse epidermis along a pathway which does not involve early prosta-glandin synthesis and phospholipid turnover and developmentof GÃ¬chalone and catecholamine refractoriness. Induction of

JANUARY 1982 345




ornithine decarboxylase activity is also not observed under invivo conditions (25), whereas in epidermal cell cultures astimulation of this enzyme by 4-O-methyl-TPA was found (40).An explanation for this discrepancy is not at hand. 4-O-Methyl-TPA-induced epidermal hyperproliferation in vivo is neither

accompanied by skin inflammation nor followed by pronouncedhyperplasia. Our results clearly distinguish the mechanism ofaction of 4-O-methyl-TPA from that of the tumor promoter TPAand lend additional support to the concept of "hyperplastic

C

I 100-

Q.O

I

iÂ«,

1O 2O 30Hours

40

Chart 5. Effect of TPA and 4-O-methyl-TPA on the activity of cyclic AMPphosphodiesterase in mouse epidermis in vivo. The phorbol esters (O O, 10nmol TPA; O O, 20 nmol TPA; â€¢ â€¢,400 nmol 4-O-methyl-TPA) weredissolved in 0.1 ml acetone each and topically applied. The animals were killedat the times indicated, and the enzyme activity was assayed in epidermishomogenate according to the procedure in Ref. 26. Points mean values of atleast 6 experiments (animals). Bars, S.D. &, average S.D. of the controls, whichreceived acetone instead of phorbol ester (24 Â±7 pmol 5'-AMP formed per mg

protein per min; n = 34).

transformation" of mouse epidermis in vivo. The term was

introduced by us in order to characterize a special kind ofproliferative response of epidermis to exogenous stimulation.Furthermore, it has been shown that this transformation istriggered by a synergistic action of the stimulus together withendogenously generated prostaglandin E2 and involves thetemporary breakdown of an endogenous control device, i.e.,the G! chalone mechanism, as well as the induction of ornithinedecarboxylase, finally leading to a rather sudden developmentof hyperplasia (24). Hyperplastic transformation seems to bealways accompanied by skin inflammation, and frequently aconcurrent desensitization of epidermis to /ÃŸ-adrenergic stimulation is observed.

The whole process of hyperplastic transformation is certainlynot due to an overshooting of epidermal cell proliferation but isthe result of a specifically induced (by prostaglandin synthesis)fundamental change of the homeostatic equilibrium of thetissue. It may be interpreted as a transient relapse of the adultepidermis into a state of differentiation and proliferation whichresembles neonatal epidermis (2, 24). One reason to call thisresponse "hyperplastic transformation" was the necessity to

distinguish it explicitly from any stimulation of normal cellproliferation in mouse epidermis. The first evidence that sucha differentiation has indeed to be made came from studies onmechanical stimulation of epidermal cell growth (2). It wasshown that, whereas epidermal damage (by means of sandpaper rubbing) induced the whole sequence of events characteristic for the development of sustained hyperplasia (withthe exception of catecholamine refractoriness), a nondamagingstimulus such as skin massage caused neither prostaglandinsynthesis (12), induction of ornithine decarboxylase (25), catecholamine (28), and chalone refractoriness nor skin inflammation (2). Although the mitotic response seen after massagewas almost indistinguishable from that after epidermal damage,damage led to epidermal hyperplasia whereas massage did not(2). It was concluded, therefore, that hyperplastic transformation is the characteristic response of adult mouse epidermis to

Table 3Tumor-promoting efficacy of 4-O-methyl-TPA as compared with TPA and RPA

Seven-week-old female NMRI mice (16 animals/group) were initiated with a single dose of 100 nmol of dimethylbenz(a)anthracene (in 0.1ml acetone). One week later, treatments with the phorbol esters (or acetone) were started. For this purpose, the compounds (in the dosesindicated) were dissolved in 0.1 ml acetone and applied twice weekly. At the end of the experiments, all animals were alive. For other details,see text.

TreatmentA.8.C.FirstTPA(4x;

10nmol)TPA(4x;10nmol)TPA(4x;10nmol)4-O-Methyl-TPA

(4x;400nmol)4-O-Methyl-TPA

(4x;400nmol)Acetone

(4x)4-O-Methyl-TPA

(4x;400nmol)TPA(4x;

10nmol)Acetone(4x)TPA(4x;

10 nmol)SecondTPA(32x;

10nmol)Acetone(32x)4-O-Methyl-TPA

(32x;400nmol)4-O-Methyl-TPA

(32x;400nmol)Acetone

(32x)4-O-Methyl-TPA

(32x;400nmol)RPA(32x;

10nmol)Acetone

(32x)RPA(32x;10nmol)RPA(32x;10 nmol)AfterRate"93000000006012

wkYield"8.1000000001.9Tumor

developmentAfterRate8930000013006715

wkYield"8.4000000.25003.3After18wkRate093071300250080Yield"8.500.120.25000.5005.2

" Percentage of tumor-bearing animals per group.

Number of tumors per animal.





injury (probably counteracting the damaging influence by building up a thicker epithelium and more resistant horny layer),whereas a nondamaging stimulus increases nothing but therate of normal chalone-controlled everyday tissue regeneration

(24).Within the framework of this concept, irritant skin mitogens

such as TPA may be regarded as compounds which eithersomehow damage the tissue or, more probably, mimic tissuedamage, whereas the nonirritant mitogen 4-O-methyl-TPA apparently resembles a nondamaging stimulus ("chemical massage"). Whether hyperplasia and skin inflammation are

causally related remains an open question. The fact that hyperplasia can be prevented by cyclooxygenase inhibition whilethe symptoms of inflammation remain unchanged is at leastinconsistent with such an assumption. Thus, skin inflammationmay be nothing more than an accompanying symptom ofhyperplastic transformation.

The observations described in this paper may also haveimportant implications for the chalone concept (for details, seeRef. 17), since chalone-controlled epidermal hyperproliferation

(which does not result in hyperplasia), previously induced onlyby skin massage, has now been repeated by means of quite adifferent mitogenic stimulus. This supports the idea of a distinctphysiological role of epidermal d chalone, i.e., as a regulatoryfactor which helps to maintain the special (nonhyperplastic)morphology and growth behavior of normal adult mouse epidermis. As yet, no exception seems to exist to the rule thathyperplastic transformation of the epidermis is preceded bychalone refractoriness, so that a causal relationship betweenboth events may exist.

As far as we know, all skin tumor promoters induce hyperplastic transformation of epidermis, whereas not every hyperplastic transformant is a skin tumor promoter. Thus, hyperplastic transformation may be regarded as necessary but certainlynot a sufficient condition of tumor promotion. As already emphasized, a suitable negative control compound for studies onphorbol ester tumor promotion should be a nonpromoting hyperplastic transformant. The fact that 4-O-methyl-TPA does not

induce hyperplastic transformation invalidates its role as anegative control compound.

In addition, 4-O-methyl-TPA is neither a potent first-stagenor a second-stage tumor promoter when tested in combinationwith either the second-stage promoter RPA or TPA. The observations of a weak activity of 4-O-methyl-TPA as a first-stagepromoter in Sencar mice (a strain highly susceptible for 2-stagecarcinogenesis) indicates that some strain differences mayexist in this respect (35). Since RPA is a strong hyperplasi-ogenic transformant, it may be concluded that hyperplastictransformation is obligatory for the second stage of promotion,probably to make the reversibly growing benign tumors generated in the classical 2-stage carcinogenesis experiment vis

ible to the naked eye (24).

ACKNOWLEDGMENTS

We thank Eva Besemfelder, Heidi Sauter, and Ingeborg Vogt for technicalassistance.

REFERENCES

1. Balmain. A., and Hecker, E. The effects of tumor promotion on 3H-choline

and 3H-glycerol incorporation into mouse epidermal phosphatidylcholine inrelation to their effect on 3H-thymidine incorporation into DMA. Z. Krebs-

forsch., 86. 251-261, 1976.

2. Bertsch, S.. Csontos, K., Schweizer. J.. and Marks. F. Effect of mechanicalstimulation on cell proliferation in mouse epidermis and on growth regulationby endogenous factors (chalones). Cell Tissue Kinet.. 9. 445-457, 1976.

3. Blumberg, P. M. In vitro studies on the mode of action of the phorbol esters,potent tumor promoters. Part 1 and 2. CRC Crit. Rev. Toxicol., 8: 153-198,199-234. 1980, 1981.

4. Boutwell, R. K. Some biological aspects of skin carcinogenesis. Prog. Exp.Tumor Res., 4: 207-250, 1964.

5. Cabot, M. C., Welsh, C. J., Callaham, M. F., and Huberman, E. Alterationsin lipid metabolism induced by 12-O-tetradecanoylphorbol-13-acetate indifferentiating human myeloid leukemia cells. Cancer Res., 40: 3674-3679,1980.

6. Castagna, M., Rochette-Egly, C., Rosenfeld, C., and Mishal, Z. Altered liquidmicroviscosity in lymphoblastoid cells treated with 12-O-tetradecanoylphor-bol-13-acetate, a tumor promoter. FEBS Lett., TOO.62-66, 1979.

7. Diamond, L, O'Brien, T. G., and Baird, W. M., Tumor promoters and the

mechanism of tumor promotion. Adv. Cancer Res., 32. 1-74. 1980.8. Fisher, P. B., Bozone, J. H., and Weinstein, I. B. Tumor promoters and

epidermal growth factors stimulate anchorage-independent growth of adenovirus-transformed rat embryo cells. Cell, 18: 695-750, 1979.

9. Fitzgerald, D. J., and Murray, A. W. Inhibition of intercellular cooperation bytumor-promoting phorbol esters. Cancer Res., 40: 2935-2937, 1980.

10. Folch, J., Lees, M., Sloane-Stanley. G. H. A simple method for the isolationand purification of total lipids from animal tissues. J. Biol. Chem., 226: 497-

509, 1956.11. Furstenberger, G., Berry, D. L., Sorg, B., and Marks, F. Skin tumor promotion

by phorbol esters is a two-stage process. Proc. Nati. Acad. Sei. U. S. A., in

press, 1981.12. Furstenberger, G., DeBravo, M., Bertsch, S., and Marks, F. The effect of

indomethacin on cell proliferation induced by chemical and mechanicalmeans in mouse epidermis in vivo. Res. Commun. Chem. Pathol. Pharmacol.,24: 533-541, 1979.

13. Grimm, W., and Marks, F. Effect of tumor-promoting phorbol esters on thenormal and the isoproterenol-stimulated level of cyclic AMP in mouse epidermis in vivo. Cancer Res., 34: 3128-3134, 1974.

14. Hecker, E. Structure-activity relationships in diterpene esters irritant andcocarcinogenic to mouse skin. In: T. J. Slaga and R. K. Boutwell (eds.),Carcinogenesis, a Comprehensive Survey, Vol. 2, pp. 11 -48. New York:Raven Press, 1978.

15. Hecker, E., Fusenig, N. E., Kunz, W., Marks, F., and Thielmann, H. W.(eds.), Carcinogenesis, A Comprehensive Survey, Vol. 7. Cocarcinogenesisand Biological Effects of Tumor Promoters. New York: Raven Press, 1982.

16. Hondius-Boldingh, W., and Laurence, E. B. Extraction, purification andpreliminary characterization of the epidermal chalone: a tissue-specificmitotic inhibitor obtained from vertebrate skin. Eur. J. Biochem., 5: 191-

198, 1968.17. Houck, J. C. (ed.). Chalones. Amsterdam: Elsevier/North-Holland, 1976.18. Kinsella, A., Mousset, S., Szpirer, C., and Radman, M. Fixation and expres

sion of recessive mutations in mammalian cells as a model for the study ofcarcinogenesis. In: P. C. Hanawalt, E. C. Friedberg, and C. F. Fox (eds.),DNA Repair Mechanisms, pp. 733-738. New York: Academic Press, 1978.

19. Kinzel, V., Richards. J., and StÃ¶hr,M. Early effects of the tumor-promotingphorbol ester 12-O-tetradecanoylphorbol-13-acetate on the cell cycle traverse of asynchronous HeLa cells. Cancer Res., 4 ): 300-305, 1981.

20. Krieg, L., KÃ¼hlmann, I., and Marks, F. Effect of tumorpromoting phorbolesters and of acetic acid on mechanisms controlling DNA-synthesis andmitosis (chalones) and on the biosynthesis of histidine-rich protein in mouseepidermis in vivo. Cancer Res., 34: 3135-3146, 1974.

21. Malaisse, W. J., Sener, A., Herchuelz, A., Carpinelli, A. R., Poloczek, P.,Winand, J., and Castagna, M. Insulinotropic effect of tumor promoter 12-O-tetradecanoylphorbol-13-acetate in rat pancreatic islets. Cancer Res., 40:3829-3831, 1980.

22. Marks, F. Isolation of an endogenous inhibitor of epidermal DNA synthesis(GÃ¬chalone) from pig skin. Hoppe-Seyler's Z. Physiol. Chem., 356. 1989-

1992, 1975.23. Marks, F. Induction of catecholamine refractoriness by isoproterenol via a

cycloheximide-sensitive reaction in mouse epidermis in vivo. FEBS Lett..113: 206-210, 1980.

24. Marks, F., Berry, D., Bertsch, S., Fischer, S. M., Furstenberger, G., andRichter, H. On the relationship between epidermal hyperproliferation andskin tumor promotion. In: E. Hecker, N. E. Fusenig, and W. Kunz (eds.),Carcinogenesis, a Comprehensive Survey, Vol. 7, pp. 331-346. New York:Raven Press, 1982.

25. Marks, F., Bertsch, S., and Furstenberger, G. Ornithine decarboxylase, cellproliferation, and tumor promotion in mouse epidermis in vivo. Cancer Res.,39. 4183-4188, 1979.

26. Marks, F., and Furstenberger, G. Effect of phorbol ester application andother mitogenic treatments on 3',5'-cyclic nucleotide phosphodiesteraseactivity in mouse epidermis in vivo. Hoppe-Seyler's Z. Physiol. Chem., 367.

1641-1650, 1980.

JANUARY 1982 347




27. Marks. F., FÃ¼rstenberger, G., and Kownatzki, E. Prostaglandin E-mediatedmitogenic stimulation of mouse epidermis in vivo by divalent cation ionophoreA 23187 and by tumor promoter 12-O-tetradecanoylphorbol-13-acetate.Cancer Res., 41: 696-702, 1981.

28. Marks, F.. Ganss, M., and Grimm, W. Agonist- and mitogen-induced desen-sitization of isoproterenol-stimulated cyclic AMP formation in mouse epidermis in vivo. Biochim. Biophys. Acta, in press, 1981.

29. Mufson, R. A., Kulkarni, P., Eakins, K. E., and Weinstein, I. B. Effects ofphorbolester tumor promoters in platelet aggregation and platelet productionof cyclooxygenase products. Cancer Res., 39: 3602-3603, 1979.

30. Mufson, R. A., Simsiman, R. C., and Boutwell, R. K. The effect of phorbolester tumor promoters on the basal and catecholamine-stimulated levels ofcyclic AMP in mouse skin and in epidermis. Cancer Res., 37: 665-669,1977.

31. Murray, A. W. Acetic acid pretreatment of initiated epidermis inhibits tumorpromotion by a phorbol ester. Experientia (Basel), 34: 1507-1508, 1978.

32. O'Brien, T. G. The induction of ornithine decarboxylase as an early, possibly

obligatory event in mouse skin carcinogenesis. Cancer Res., 36: 2644-

2653, 1976.33. Rochette-Egly. C., Chouroulinkov, I., and Castagna. M. Cyclic nucleotide

levels in rat embryo fibroblasts treated with tumor-promoting phorbol diester.J. Cyclic Nucleotide Res., 5: 385-395, 1979.

34. Schultz, R. M.. Chirigos, M. A., and Olkowski, Z. L. Stimulation and inhibitionof neoplastic cell growth by tumor promoter-treated macrophages. Cell.

Immunol., 54: 98-105, 1980.35. Slaga, T. J., Fischer, S. M., Nelson, K., and Gleason, G. L. Studies on the

mechanism of skin tumor promotion: evidence for several stages of promotion. Proc. Nati. Acad. Sei. U. S. A., 77: 3659-3663, 1980.

36. Slaga, T. J., Sivak, A., and Boutwell, R. K. (eds.). Carcinogenesis. AComprehensive Survey, Vol. 2. New York: Raven Press, 1978.

37. Verma, A. K., Froscio, M., and Murray, E. A. Croton oil- and benzo(a)pyrene-induced changes in cyclic AMP and cyclic GMP phosphodiesterase activitiesin mouse epidermis. Cancer Res., 36: 81 -87. 1976.

38. Yamasaki, H. Reversible inhibition of cell differentiation by phorbol esters asa possible mechanism of the promotion step in chemical carcinogenesis. In:R. Montesano, H. Bartsch, and L. Tomatis (eds.), Molecular and CellularAspects of Carcinogen Screening Tests, IARC Scientific Publications No.27 pp. 91-112. Geneva: World Health Organization, 1979.

39. Yotti, L. P., and Trosko, J. E. Inhibition of metabolic cooperation in mammalian tissue culture cells by phorbol myristate acetate. In Vitro, (Rockville),15: 208-209, 1979.

40. Yuspa, S. H., Lichti, U., Ben, T., Patterson, E., Hennings, H., Slaga, T. J.,Colburn, N., and Kelsey, W. Phorbol esters stimulate DNA synthesis andornithine decarboxylase activity in mouse epidermis in epidermal cell cultures. Nature (Lond.), 262. 402-404, 1976.

41. Zur Hausen, H., Bornkamm, G. W., Schmidt, R., and Hecker, E. Tumorinitiators and promoters in the induction of Epstein Barr virus. Proc. Nati.Acad. Sei. U. S. A., 76. 782-785, 1979.




1982;42:342-348. Cancer Res Gerhard Fürstenberger, Hartmut Richter, Thomas S. Argyris, et al. Promotersin Studies on the Biological Effects of Phorbol Ester TumorEvidence for Its Uselessness as a Negative Control Compound

:in Vivo-Tetradecanoylphorbol-13-acetate on Mouse Skin O-Methyl-12-OEffects of the Phorbol Ester 4-

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