Effects of 3-Lsobutyl-1-Methylxanthine and Cyclic Nucleotides on 12-0

[CANCER RESEARCH 41, 3918-3926, October 1981] 0008-5472/81 /0041-0000$02.00 Effects of 3-lsobutyl-1-methylxanthine and Cyclic Nucleotides on 12-0- Tetradecanoylphorbol-13-acetate-induced Ornithine Decarboxylase Activity in Mouse Epidermis in Vivo1

Jean-Pierre Perchellet and R. K. Boutwell

McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706

ABSTRACT INTRODUCTION

A single topical application of 8.5 nmol of 12-O-tetradeca- In the 2-step model system for the production of skin tumors noylphorbol-13-acetate (TPA) to mouse skin induced rapid and in mice, repeated topical applications of the potent tumor transient increases in the levels of both guanosine cyclic 3':5'- promoter TPA2 to initiated skin led to the formation of skin monophosphate and adenosine cyclic 3':5'-monophosphate, papillomas and carcinomas (7). The molecular basis for the followed by the stimulation of ornithine decarboxylase action of TPA in mouse epidermis is not understood. Most of (ODC, L-ornithine carboxy-lyase, EC 4.1.1.17) activity. Peak the early biochemical responses observed following TPA treat syntheses of guanosine cyclic 3':5'-monophosphate and aden ment (reviewed in Ref. 7), particularly the stimulation of poly- osine cyclic 3':5'-monophosphate were achieved within 10 and amine synthesis (6, 37, 38), have been proposed to be nec 60 min, respectively, while ODC activity was maximally stimu essary components of the promotion process. However, little lated between 4.5 and 6 hr. The increased levels were dose is known about the biochemical mechanism by which signals dependent and correlated with the tumor-promoting abilities of resulting from TPA contact with the plasma membrane could diverse compounds. However, the magnitudes of the cyclic be transmitted into the interior of the epidermal cell to induce nucleotide and ODC responses to TPA were quite different (3- gene expression. to 7- and 70-fold, respectively). In other systems, an increasing body of literature indicates A single topical application of 10 jumol of 3-isobutyl-1-meth- that ODC activity may be regulated via stimulation of a cyclic ylxanthine (IBMX), which was able to raise the levels of cyclic nucleotide-dependent process (reviewed in Ref. 47). The pres nucleotides almost as much as did TPA, produced only a 9- ence of active adenylate cyclase, guanylate cyclase, phospho- fold increase in ODC activity. IBMX, as well as other phosphodiesterases, and protein kinases has been shown in the epi diesterase inhibitors, enhanced the magnitude and the duration dermis (26, 32, 53, 59). The possibility that some of the effects of the increases in cyclic nucleotide levels and ODC activity of TPA might be mediated by phosphorylation of cellular com produced by TPA. Maximum stimulation of TPA-induced ODC ponents through a cyclic nucleotide-dependent process is quite activity was achieved when IBMX was applied within the 30- attractive. Although conflicting data have been reported con min time interval preceding TPA treatment. As a result of the cerning early changes in cyclic nucleotide levels after TPA IBMX pretreatment, the same or higher levels of cyclic nucleo treatment (reviewed in Ref. 41), regulation of the enzymes tides and ODC activity could be induced with about 10 times controlling the metabolism of the cyclic nucleotides by TPA has less TPA or with weak and moderate tumor promoters, as been described (32, 34, 45, 53, 57). Recently, investigations compared with the increased levels attributable to TPA alone. in this laboratory demonstrated that TPA did stimulate rapidly However, in the presence of IBMX, a discrepancy was still the production of cyclic GMP and cyclic AMP in vitro, using observed between the magnitude of the increased cyclic nu suspensions of freshly isolated epidermal cells (41). In the cleotide response to TPA (6- to 14-fold) and the 150-fold same system, time- and dose-dependent relations were ob induction of ODC activity produced by TPA. served between TPA-increased cyclic nucleotide levels and In addition, single or combined topical treatments with gua ODC activity. Moreover, the phosphodiesterase inhibitor IBMX nosine cyclic 3':5'-monophosphate and adenosine cyclic 3': and the adenylate cyclase stimulator cholera toxin that raised 5'-monophosphate were unable to mimic the effect of TPA on cyclic AMP levels also induced ODC activity and further en ODC activity and even depressed significantly basal and TPA- hanced the stimulation of ODC activity produced by TPA (41). induced ODC activities in the presence of IBMX. Therefore, the In the present study, the magnitude, the duration, and the findings presented in this and an accompanying paper suggest specificity of the increases in cyclic nucleotide levels and ODC that, in mouse epidermis in vivo, if TPA-increased cyclic nu activity were determined in mouse epidermis in vivo at early cleotides participate in the molecular mechanism by which stages following topical treatment of mouse.skin with IBMX, ODC activity is induced, their controlled degradation may be cyclic nucleotides, and/or agents of varying tumor-promoting equally important. abilities. New evidence is provided that alterations in the mag-

' This material was presented in part at the Annual Meeting of the American 2 The abbreviations used are: TPA. 12-O-tetradecanoylphorbol-13-acetate; Association for Cancer Research, San Diego, Calif., May 1980 (42). This inves ODC. ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17); cyclic tigation was sponsored by Grants CA-07175 and CA-22484 from the NIH. This GMP, guanosine cyclic 3':5'-monophosphate; cyclic AMP, adenosine cyclic 3': is Paper 1 in a series exploring the role of cyclic nucleotides in the mechanism 5'-monophosphate; IBMX, 3-isobutyl-1-methylxanthine; PDD, phorbol-12,13-di- of action of TPA. Ref. 43 is the following paper in this series. decanoate; PDB. phorbol-12,13-dibenzoate; PDA, phorbol-12,13-diacetate; Received March 18. 1981; accepted July 15. 1981. DMBA, 7,12-dimethylbenz[a]anthracene.

3918 CANCER RESEARCH VOL. 41

nitude and the duration of the early and transient elevation of ment, the mice were killed by cervical dislocation and epidermis from individual mice was separated by a brief heat treatment (57Â° for 30 cyclic GMP and cyclic AMP observed after TPA may modulate the effect of the tumor promoter on ODC activity. sec) as described previously (27). The epidermal preparations from 2 mice were pooled in 2 ml of 25 mM Tris-HCI buffer, pH 7.6, containing 4 mM dithiothreitol, 1 mM EDTA, and 0.2 mM pyridoxal 5'-phosphate, MATERIALS AND METHODS homogenized with a Polytron PT 10 homogenizer for 10 sec at Setting 6, and centrifuged at 30,000 x g for 30 min to give a soluble Chemicals. TPA, mezerein, FDD, PDB, PDA, and phorbol were all supernatant (38). ODC activity was determined in 0.2-ml aliquots of the clear supernatants by measuring the release of 14CO2 from DL- purchased from Peter Borchert, Chemical Carcinogenesis (Eden Prai rie, Minn.), and DMBA was from Eastman Organic Chemicals (Roch [1-14C]ornithine hydrochloride as described previously (54). The sub ester, N. Y.). Cyclic AMP, cyclic GMP, aminophylline, caffeine, papav strate concentration routinely used was 0.4 mM L-ornithine hydrochlo erine, theophylline, crystalline bovine serum albumin, and L-ornithine ride containing 0.5 /iCi of oi_-{1-14C]ornithine hydrochloride. The assays hydrochloride were obtained from Sigma Chemical Co. (St. Louis, Mo.), were carried out in duplicate, and all values were corrected against no and IBMX was from Aldrich Chemical Co. (Milwaukee, Wis.). Cyclic enzyme or boiled enzyme blanks; ODC activity was expressed as nmol [3H]GMP (39.9 Ci/mmol), cyclic [14C]AMP (40 mCi/mmol), and Di_-[1- of CO; released in 60 min per mg of protein. 14C]ornithine hydrochloride (52.8 mCi/mmol) were purchased from DNA and Protein Determinations. The acid-insoluble pellet obtained New England Nuclear (Boston, Mass.). Ion-exchange resins were ob after cyclic nucleotide extraction was washed twice with 2 ml of ice- tained from Bio-Rad (Rockville Center, N. Y.). Calf thymus DNA was cold 0.5 N perchloric acid and once with 2 ml of 100% ethanol, and from Calbiochem-Behring Corp. (La Jolla, Calif.). All other chemicals the DNA was hydrolyzed in 2 ml of 0.5 N perchloric acid for 10 min at 90Â°. DNA content in the clear supernatant was determined by the were reagent grade and were obtained commercially. Treatment of Mice. Female Charles River CD-1 mice (Wilmington, diphenylamine procedure of Burton (8) with calf thymus DNA as the Mass.), housed in screen-bottomed stainless steel cages in light- and standard. The protein concentration of the epidermal extracts was temperature-controlled rooms with food and water ad libitum, were assayed with Bio-Rad dye reagent, using crystalline bovine serum used for experimentation when 7 to 9 weeks old. The dorsal skin of the albumin as the standard. mice was shaved at least 2 days before treatment, and only those mice showing no hair regrowth were used for experimentation. The solutions of mezerein, phorbol, the 4 phorbol diesters, and DMBA were prepared RESULTS in acetone and delivered to the shaved backs of individual mice in a volume of 0.2 ml. The phosphodiesterase inhibitors used for these Effects of IBMX on the Time Courses for the Increases in experiments, dissolved in 96% acetone-4% water, were applied to Cyclic Nucleotide Levels and ODC Activity by TPA. A single mouse skin in 0.2-ml volumes, with the exception of the dose of 5 /xmol topical application of 8.5 nmol of TPA to mouse skin induced of papaverine which was given in 2 consecutive applications of 0.2 ml, rapid increases (3- to 7-fold) in the levels of epidermal cyclic each with 2.5 jimol. The doses of 0.25 /Â«noiof cyclic AMP or 0.16 GMP and cyclic AMP relative to DNA content within the 120- Â»mol of cyclic GMP were delivered topically to mouse skin in 2 min time interval studied. As shown in Chart 1, the level of consecutive volumes of 0.2 ml of 96% acetone-4% water, each con cyclic GMP increased within 2 min of TPA treatment, reaching taining 0.125 or 0.08 /imol, respectively. Multiple treatments were a peak at 10 min and declining to the basal level by 60 min. In administered at twice-weekly intervals; the mice were given the phos contrast, the cyclic AMP level was increased 10 min after TPA phodiesterase inhibitors and/or the cyclic nucleotides immediately treatment and continued to rise until 60 min before declining. before and to the same area of skin as the application of TPA or the other agents of varying tumor-promoting abilities. In every experiment, Acetone treatment did not induce any significant change in the all mice received the same volume of solvent. basal levels of either cyclic nucleotide over the course of the Determination of Cyclic GMP and Cyclic AMP Levels. At the experiment. As reported previously (6, 37, 38), epidermal ODC designated time after treatment, the mice were killed by cervical activity was maximally induced (70-fold) between 4 and 6 hr dislocation and immediately frozen flat on solid CO2. After immersion following TPA treatment and returned to basal levels beyond a for 15 sec in liquid N., the epidermis were scraped off with a surgical 12-hr period (Chart 2). blade, and the scrapings from one mouse were immediately homoge Treatment of the same area of skin with 10 jumol of IBMX nized in 5 ml of ice-cold 5% trichloroacetic acid with a Polytron PT 10 elicited a slow but steady accumulation of cyclic GMP and homogenizer for 3 x 15 sec at Setting 6. Trace amounts of cyclic [3H]GMP and cyclic [I4C]AMP (each with 5000 dpm) were added to the cyclic AMP (Chart 1). The effect of IBMX seemed to fade beyond 60 min. In contrast with TPA, the 3- to 5-fold increases samples to correct for the recoveries of the 2 cyclic nucleotides, and in cyclic nucleotide levels produced by IBMX were accompa the homogenates were processed as described previously (41 ). Briefly, cyclic AMP and cyclic GMP were purified from the ether-extracted nied by only a 9-fold enhancement of basal ODC activity at 5 acid-soluble samples on 8- x 0.5-cm Dowex AG 1-X8 columns (200 to hr (Chart 2). 400 mesh) in the formate form. After the above column was washed Topical application of IBMX immediately before TPA en with 10 ml of 0.1 N formic acid, complete separation of the 2 cyclic hanced (about 2-fold) the magnitude of both the cyclic nucleo nucleotides was achieved by sequential elution with 7 ml of 2 N formic tide and the ODC responses to TPA (Charts 1 and 2, respec acid and 7 ml of 4 N formic acid (33), as assessed by counting aliquots tively) without altering the general time courses observed pre of the 2 fractions for 3H and 14Cdpm of the tracers. Recovery of each viously. However, as a result of the IBMX pretreatment, the cyclic nucleotide was at least 75%. After lyophilization, the cyclic AMP- peak levels of cyclic GMP, cyclic AMP, and ODC activity were and cyclic GMP-containing samples were reconstituted in 1 ml of 50 mM sodium acetate buffer, pH 6.2. The cyclic AMP (50) and succiny- broadened so that the same or higher levels of cyclic nucleo lated cyclic GMP (16) contents were determined by radioimmunoassay, tides, compared with the response to TPA alone, were now using the reagents, antigens, and antibodies supplied by Collaborative maintained for about 90 min longer, and the ODC activity Research, Inc. (Waltham, Mass.). Under these conditions, the assays exceeded that induced by TPA alone through the period from of cyclic AMP and cyclic GMP were highly sensitive and specific; 2.5 to 9 hr. cross-reactivity of even closely related nucleotides was minimal. Effects of the Dose and Time of Treatment with IBMX on Determination of ODC Activity. At the appropriate time after treat the Induction of ODC Activity by TPA. As shown in Table 1,

OCTOBER 1981 3919

interval following TPA treatment to measure nearly peak levels of cyclic GMP and cyclic AMP in the presence and absence of IBMX. As shown in Chart 4, both cyclic GMP and cyclic AMP levels, as well as ODC activity, were stimulated in the same dose-dependent manner by TPA. The lowest dose of TPA eliciting a significant increase in both cyclic nucleotide levels and ODC activity was 0.07 nmol; maximal levels occurred with 5.66 and 17 nmol of TPA. The effect of each dose of TPA tested was enhanced after IBMX treatment (Chart 4). Therefore, as a result of the IBMX treatment, a dose of TPA about 10 times lower than that routinely used in a single treatment was sufficient to induce

2 10 30 60 120 Time (minutes) Chart 1. Time-response curves showing the effects of IBMX on basal and TPA-stimulated cyclic GMP (A) and cyclic AMP (ÃŸ)levels in mouse epidermis in vivo. The mice were treated topically with acetone plus acetone (O), 10 /Â«noiof IBMX plus acetone O, acetone plus 8.5 nmol of TPA (â€¢).or 10 fimol of IBMX plus 8.5 nmol of TPA (A). The solutions were applied to the skin in 0.2-ml aliquots in close time sequence, and the mice were killed for cyclic GMP and cyclic AMP determination at the indicated times following treatment (Time 0). Each point o 2 6 8 10 12 14 16 18 24 represents the mean of duplicate determinations of cyclic GMP, cyclic AMP, and i ime (hours) DNA from individual epidermal extracts prepared from 6 mice in 2 different Chart 2. Time-response curves showing the effect of IBMX on basal and TPA- experiments. Bars, S.O. Basal cyclic GMP (16 Â±2 fmol/>g DNA) and cyclic AMP induced ODC activities in mouse epidermis in vivo. The mice were treated (0.25 Â±0.05 pmol//ig DNA) levels at Time 0 have been subtracted from the topically with 10 Â»molof IBMX plus acetone (O), acetone plus 8.5 nmol of TPA results. The time is plotted on a logarithmic scale, a, results not significant versus (â€¢),or 10 /imol of IBMX plus 8.5 nmol of TPA (D). The solutions were applied to acetone plus acetone; b. p < 0.0005, significantly greater versus acetone plus the skin in 0.2-ml aliquots in close time sequence, and the mice were killed for acetone; c. results not significant versus IBMX plus acetone; d, p < 0.01, enzyme assay at the indicated times following treatment (Time 0). Each point significantly greater versus IBMX plus acetone. represents the mean of duplicate determinations of enzyme activity from 6 groups of mice in 2 different experiments; each group contained the combined soluble epidermal extracts prepared from 2 mice. Bars, S.D. Basal ODC activities in stimulations of basal and TPA-induced ODC activities by topical control mice treated with acetone plus acetone (0.125 Â±0.018 nmol CO.-) have been subtracted from the results at each time point, a, p < 0.0005, significantly applications of IBMX were dose dependent. The induction of greater versus acetone plus acetone; b, results not significant versus acetone ODC activity became apparent with a dose of 0.5 /imol of IBMX plus acetone; c, p < 0.0005, significantly greater versus acetone plus TPA; d, results not significant versus acetone plus TPA. and was stimulated maximally at 10 to 20 jumol of IBMX. The effects of IBMX and TPA were clearly more than additive. The stimulation of TPA-induced ODC activity was dependent Table 1 Effect of the dose of IBMX on basal and TPA-induced ODC activities in mouse on the time of IBMX application. Maximal stimulation occurred epidermis in vivo when IBMX was given concurrently with TPA or 30 min before The conditions of the experiment were identical with those in Chart 2. Groups TPA application. Effectiveness was rapidly lost at treatment of mice were treated topically with 0.1 to 20 /imol of IBMX immediately before times further from the time of application of TPA (Chart 3), so application of acetone or 8.5 nmol of TPA. The mice were killed 5 hr later for determination of ODC activity. that the ability of IBMX to potentiate the effect of TPA was ODC activity (nmol CO. in 60 min/mg protein) reduced by more than 50% when it was applied to mouse skin 2 hr before or after the treatment with TPA. This is in accord TreatmentAcetone acetone0.115 TPA (8.5nmol)4.432 with the observation that IBMX becomes ineffective in prevent Â±0.019a Â±0.310 IBMX (0.1 .ni...!) 0.125 Â±0.0186 4.601 Â±0.320C ing the degradation of epidermal cyclic nucleotides beyond 60 5.067 Â±0.358Â° IBMX (0.5 ..mol) 0.164 Â±0.022d min of its application to the skin. However, addition of TPA 4 to IBMX (1 /imol) 0.198 Â±0.037 5.664 Â±0.452 6 hr after IBMX treatment, at a time when basal ODC activity IBMX (2.5 /.mol) 0.261 Â±0.050 7.357 Â±0.685 was maximally stimulated by the IBMX pretreatment (Chart 2), IBMX (5 /.mo!) 0.392 Â±0.060 9.312 Â±0.708 IBMX {I I.)nmol) 0.467 Â±0.068 13.865 Â±1.040 resulted in a slight but significant increase of the ODC response IBMX (15 .imol) 0.487 Â±0.090 14.620 Â±1.211 to TPA. IBMX (20 nmol)+ 0.494 Â±0.086+ 14.710 Â±1.180 Effects of IBMX on the Dose-dependent Stimulations of Mean Â±S.D. " Results not significant versus acetone plus acetone. Cyclic Nucleotide Levels and ODC Activity by TPA. In order ' Results not significant versus acetone plus TPA. to determine the levels of both cyclic nucleotides in the same d p < 0.005, significantly greater versus acetone plus acetone. epidermal extracts, 20 min was selected as the best time 8 p< 0.01. significantly greater versus acetone plus TPA.

3920 CANCER RESEARCH VOL. 41

is not a phorbol derivative, is much less able than TPA to promote tumor formation but is very effective in Stage II pro motion (30,49). However, mezerein caused increases in cyclic nucleotide levels and ODC activity comparable with those I I l4 induced by TPA. A single topical application of 10 /tmol of IBMX enhanced by about 2-fold the stimulation of cyclic nu ; i2 cleotide levels and ODC activity produced by each of the above E compounds (Chart 6). Moreover, after IBMX pretreatment, the Ã¬ IO levels of cyclic nucleotides and ODC activity produced by the O weak or moderate tumor promoters were boosted to values 1 8 corresponding to the effects of the strong promoting agents in !" 6 the absence of IBMX. For instance, the moderate tumor pro ~ "o moter PDD was able, in the presence of IBMX, to increase the EE Â°â€”4 levels of cyclic GMP, cyclic AMP, and ODC activity as much as the powerful TPA did alone. -12 -IO -8 -6 -4 -2 *2 Effects of Other Phosphodiesterase Inhibitors. Not only Time (hours) Chart 3. Effect of the time of application of a single dose of IBMX on TPA- induced ODC activity in mouse epidermis in vivo. Groups of mice were treated topically with 10 /iirtol of IBMX at various times before or after application of 8.5 nmol of TPA (Time 0). The mice were killed 5 hr after TPA treatment for determination of ODC activity. The conditions of the experiment were identical with those in Chart 2. ODC activity in control mice treated with acetone plus 8.5 20 nmol of TPA only was 4.832 Â±0.365 nmol CO2 (hatched area). Bars, S.D. a, O gÃ¬ results not significant versus acetone plus TPA; b,p< 0.005, significantly greater o.?- versus acetone plus TPA. 20 same or higher levels of cyclic GMP, cyclic AMP, and ODC activity. For instance, in the presence of IBMX, 1.89 nmol of TPA increased cyclic nucleotide levels 7- to 11-fold at 20 min and induced ODC activity 74-fold at 5 hr, thus exceeding the 5-z I.6 effects of 17 nmol of TPA alone. Â° Effects of Multiple Applications of IBMX and TPA on In duction of Epidermal ODC Activity. Development of skin tu mors requires repeated applications of a tumor promoter to Â¿ios initiated skin. Furthermore, it has been shown previously that repeated applications of TPA led to much greater degrees of induction of ODC activity than those observed after the first treatment (38, 56). Therefore, it is of interest to determine if the phosphodiesterase inhibitor IBMX is able to further enhance 12 the greater induction of ODC activity produced by repeated TPA treatments. The levels of ODC induction were determined following multiple applications of IBMX plus TPA to mouse skin initiated with DMBA. The results shown in Chart 5 indicate that the degree of stimulation of ODC activity by TPA was increased about 2-fold after the second TPA treatment and plateaued at about 2.5-fold between the sixth and tenth applications of TPA, as compared with the response to the first TPA treatment. IBMX, which has been shown to enhance the induction of ODC 0.02 0.21 1.89 17.00 activity produced by a single application of TPA, was able to TPA (nmol) further increase the greater ODC responses elicited by each Chart 4. Effects of a single topical application of IBMX on the dose-dependent successive promoter treatment. production of cyclic GMP (A) and cyclic AMP (8) and on induction of ODCactivity Effects of IBMX on the Cyclic Nucleotide and ODC Re (C) in response to TPA In mouse epidermis in vivo. Groups of mice were treated sponses to Compounds of Varying Tumor-promoting Abili with acetone (O) or 10 /imol of IBMX (â€¢)immediatelybefore application of 0.02 to 17 nmol of TPA. The mice were Killed 20 min later for cyclic GMP and cyclic ties. Within a series of agents of varying tumor-promoting AMP determination or after 5 hr for enzyme assay. The conditions of the potencies, the magnitude of the increases in cyclic nucleotide experiments were identical with those in Charts 1 and 2. Basal cyclic GMP and cyclic AMP levels in control mice treated with acetone plus acetone (10 Â±1 levels and ODC activity correlated with their promoting abilities fmol.'mi DMAand 0.19 Â±0.03 pmol/pg DNA, respectively) or 10 /

OCTOBER 1981 3921

activation of epidermal cyclic nucleotide phosphodiesterases, combined treatments including IBMX, cyclic GMP, and cyclic 35 AMP were applied. In the presence of IBMX, cyclic GMP and cyclic AMP treatments were again unable to reproduce the 30 activity of TPA and very significantly reduced the enhancement of basal and TPA-induced ODC activities caused by IBMX

ftÂ« alone. Moreover, incubation of isolated epidermal cells (41) with 1 /IM to 1 HIM cyclic GMP, cyclic AMP, or their dibutyryl lÃ derivatives resulted in a concentration-dependent inhibition of 20 Si basal and TPA-induced ODC activities, whether or not 0.5 mw f o IBMX was present in the incubation medium (data not shown). 15 A dye exclusion test performed at the end of each incubation period revealed no change in cell viability, and normal ODC S - IO induction by TPA was restored following replacement of the l E cells in cyclic nucleotide-free medium. The same doses of sodium butyrate did not elicit any modification of basal and TPA-induced ODC activities.

2466 IO Number of Applications of TPA DISCUSSION

Chart 5. Effect of repeated topical applications of IBMX and TPA on induction Recent evidence of sequential oscillations in the levels of of ODC activity in mouse epidermis in vivo. Groups of mice were initiated with 0.1 muni of DM BA in 0.2 ml of acetone: 2 weeks after initiation, the mice were both cyclic GMP and cyclic AMP induced by TPA (22, 41, 46) treated twice a week for the duration of the experiment with either acetone (O) or but not by nonpromoting phorbol esters (46) has been reported 10 Â»inniof IBMX (A) immediately before application of 8.5 nmol of TPA. The mice were killed 5 hr after the indicated number of applications of TPA for in vitro. In mouse epidermis (36, 58) and various cell systems enzyme assay. The conditions of the experiment were identical with those in (15, 51), it is noteworthy that cyclic GMP levels are increased Chart 2. ODC activities in control mice treated repeatedly with acetone plus acetone or 10 jimol of IBMX plus acetone have been subtracted from the appropriate values. Bars. S.D. a, results not significant versus second application of acetone plus TPA; p < 0.005, significantly greater versus first application of acetone plus TPA. b. results not significant versus second application of IMBX plus TPA.

IBMX but a variety of other phosphodi este rase inhibitors as well were able to increase basal and TPA-stimulated cyclic nucleotide levels and ODC activities (Chart 7). Their effective ness as stimulators of the cyclic nucleotide and ODC responses to TPA was as follows. Papaverine and IBMX appeared to be the most effective, followed by caffeine and aminophylline, with theophylline having a much lesser effect. Although papaverine- and IBMX-increased cyclic nucleotide levels were comparable to those produced by TPA, the ODC inductions that resulted from papaverine and IBMX treatments were 7 to 9 times smaller than the ODC response to TPA. In the presence of the above compounds, the degree of stimulation of ODC activity by TPA was also much greater than that of the cyclic nucleotides. Effects of Cyclic Nucleotides on Basal and TPA-induced ODC Activities. If the TPA-increased levels of cyclic nucleo tides play some role in the regulation of ODC (41 ), then eleva tions in epidermal cyclic GMP and cyclic AMP produced in vivo by phosphodiesterase inhibitors and/or exogenously added cyclic nucleotides should mimic the enzymic induction caused by the tumor promoter. As shown in Table 2, topical applica tions of cyclic GMP or cyclic AMP to mouse skin were totally ineffective in raising, and even slightly depressed, basal and TPA-stimulated ODC activities; additive inhibitory effects were Chart 6. Effect of IBMX treatment on the levels of cyclic GMP (A) and cyclic observed when the 2 cyclic nucleotides were applied together. AMP (B) and on the induction of ODC activity (C) in response to compounds of No change in ODC activity was observed when the cyclic varying tumor-promoting abilities in mouse epidermis in vivo. Groups of mice were treated with acetone (shaded columns') or 10 /Â»molofIBMX (open columns) nucleotides were applied 4 hr prior to the acetone, TPA, or immediately before topical application of 8.5 nmol of the indicated compounds IBMX plus TPA treatments (data not shown), suggesting that (or acetone control). Bars, S.D. The mice were killed 20 min or 5 hr later for the enzyme inhibition by cyclic GMP and cyclic AMP was not cyclic nucleotide determinations and enzyme assay, respectively. The conditions of the experiments were identical with those in Charts 1 and 2. a, results not caused by a local toxic effect. Since applications of large significant versus acetone plus acetone; b. results not significant versus IBMX amounts of exogenous cyclic nucleotides could trigger the plus acetone.

3922 CANCER RESEARCH VOL. 41

of cyclic nucleotides. At physiological concentrations of sub 80 strate, the rate of cyclic GMP hydrolysis by cytoplasmic phos phodiesterase is usually greater than that of cyclic AMP (26). 60 Therefore, after TPA treatment, newly synthesized cyclic GMP L may simply be degraded much faster than is cyclic AMP, aio â€ž¿ L. "40 resulting in a cyclic AMP level that is still increasing while cyclic GMP is declining. Epidermal phosphodiesterase activity, which Si may be induced as early as 10 min after TPA treatment as 20 suggested by our data, has been demonstrated to reach higher activities later (32, 53), thus allowing the degradation of the accumulated cyclic AMP. .B l It is difficult to pinpoint the reasons why, in an earlier work, no significant change in cyclic nucleotide levels could be detected after TPA treatment using epidermal-dermal prepa rations (29). The inability to measure increased level of cyclic u E nucleotides does not mean that they have not occurred. The use of a potent phosphodiesterase inhibitor (IBMX) that en !Ã®"C hanced the magnitude and the duration of the cyclic nucleotide response to TPA was particularly helpful in our studies in vitro < s (41) and in vivo, since epidermal cyclic GMP and cyclic AMP seem to be rapidly synthesized and rapidly turned over after S.E TPA treatment. However, we did not determine the possible changes of cyclic nucleotide levels in the extracellular com i partment that could result from cyclic nucleotide excretion from r* the cells. In our experiments, the levels of epidermal cyclic GMP and cyclic AMP, expressed per/^g of DMA, are in the fmol and pmol ranges, respectively. Since 2 x 106 isolated epider mal cells contain about 10 jug of DNA, the levels of cyclic Chart 7. Effects of diverse phosphodiesterase inhibitors on basal and TPA- nucleotides measured in vivo appear to be about 5 times lower stimulated cyclic GMP (A) and cyclic AMP (B) levels and on ODC activities (C) in than those assayed in vitro (41). Furthermore, in vitro, the mouse epidermis in wVo.Groups of mice were treated with 5 /imol of the indicated compounds immediately before topical application of acetone (shaded columns) changes in cyclic nucleotide levels are determined in a rather or 8.5 nmol of TPA (open columns'!. Bars, S.D. The mice were killed 20 min or 5 purified epidermal preparation containing a high ratio of active hr later for cyclic nucleotide determinations and enzyme assay, respectively. The and TPA-responsive isolated basal cells (41), whereas in vivo conditions of the experiments were identical with those in Charts 1 and 2. a. p < 0.005, significantly greater versus acetone plus acetone; b, p < 0.005, the variations of cyclic nucleotide levels are determined in significantly greater versus acetone plus TPA. scrapings of epidermis with probably a lower ratio of basal cells due to the presence of all other more differentiated and within minutes of TPA treatment. However, the effect of TPA keratinized cell types. In addition, we were obliged to compro on cyclic AMP is more controversial (4, 21, 31, 36, 53, 55). mise for an experimental time interval of 20 min after TPA Other reports concerning the impaired balance of cyclic nu- treatment, giving near-maximal levels for both cyclic GMP and cleotides at 6 (44), 12(19), and 36 (5) hr after single or multiple (17) treatments with TPA, when many TPA-induced phenomena Table 2 Effects of topical applications of IBMX, cyclic GMP, and cyclic AMP on basal have already been maximally stimulated, reflect the high ratio and TPA-induced ODC activities in mouse epidermis in vivo of cyclic GMP to cyclic AMP characteristic of benign and Groups of mice were treated with the indicated compounds immediately before application of acetone or 8.5 nmol of TPA. The reagents were prepared as malignant proliferative skin diseases (59) but do not preclude indicated in "Materials and Methods" and were added in the stated sequence. the involvement of cyclic nucleotides at earlier stages of the The doses used were 0.25 /

OCTOBER 1981 3923

cyclic AMP (Chart 1). Therefore, it is obvious that the values maintained for a longer period of time after IBMX plus TPA reported in Charts 4, 6, and 7 represent only 70 to 80% of the treatment, suggesting that IBMX, by inhibiting or slowing down phenomenon studied, as compared with the values of cyclic temporarily the degradation of newly synthesized cyclic GMP GMP and cyclic AMP that could have been obtained at 10 and and cyclic AMP, increased the effectiveness of the tumor 60 min, respectively. promoters as stimulators of the production of cyclic nucleo The exact role of this early and transient increase in cyclic tides. In accord with our previous findings in vitro (41), IBMX GMP and cyclic AMP levels in the mechanism of action of the pretreatment also enhanced the magnitude of the ODC re tumor promoters remains to be determined. ODC activity has sponse to the diverse tumor promoters tested in vivo. In the been induced in vivo and in vitro by raising the extracellular presence of IBMX, only one-tenth the quantity of TPA was concentration of cyclic nucleotides (reviewed in Ref. 41) or by required to induce both peak levels of cyclic nucleotides and elevating intracellular cyclic nucleotides with the use of com maximally induced ODC activity as compared with the effects pounds known to affect the activity of the enzymes controlling of TPA alone. However, even in the presence of the phospho the metabolism of the cyclic nucleotides such as phosphodi- diesterase inhibitors, TPA and the other tumor promoters tested esterase inhibitors (1, 9,10,12, 23, 41, 48), cholera toxin (35, still caused disproportionate increases in cyclic GMP and cyclic 41), prostaglandins (1, 23, 40), adrenergic agents (1, 52), and AMP compared to the magnitudes of their induction of ODC growth factors and diverse hormones (reviewed in Ref. 24). activity. Furthermore, sequential increases in cyclic nucleotide levels, In the experiment depicted in Table 1, treatments with 10 cyclic nucleotide-dependent protein kinase, ODC, and RNA jumol of IBMX or 8.5 nmol of TPA raised ODC activity from a polymerase I activities have been described (2, 14, 25, 28, 39, basal value of 0.115 nmol COz in 60 min per mg protein to 47). In quiescent cultures of Chinese hamster ovary cells, the stimulated levels of 0.467 and 4.432 nmol CO2, respectively. major action of cyclic AMP during the initial 2 hr of the induction However, when these compounds were added together, they of ODC activity by serum is thought to be the activation of type did not raise the ODC activity to a value of only 4.899 nmol I cyclic AMP-dependent protein kinase which then may phos- CO2 but to a more than additive activity of 13.865 nmol CO2. phorylate nuclear proteins, which in turn enhance the transcrip- Therefore, the synergistic effects of IBMX and TPA on ODC tional process leading to the induction of ODC (13). Based on induction cannot be explained solely on the basis of increased results obtained in vitro, a recent report suggested that cyclic cyclic nucleotide levels. Our data indicate that TPA seems to nucleotides might play a mediatory role in the regulation of have an effect distinct from the cyclic nucleotides which dra epidermal ODC activity by TPA (41). In the present study, matically induces ODC. Separate routes for the induction of however, although peak levels of cyclic GMP and cyclic AMP ODC activity by aminophylline and triiodothyronine involving precede the increase in ODC activity when TPA is topically cyclic AMP-mediated events or direct nuclear interaction have applied to mouse skin, the degree of stimulation of the cyclic also been suggested (11 ). Whether the enhancement of basal nucleotides and that of ODC activity are not proportional. and TPA-induced ODC activities by phosphodiesterase inhibi To enhance basal and TPA-increased cyclic nucleotide levels tors is entirely due to an elevation of cyclic nucleotides is in mouse epidermis in vivo, single or multiple topical treatments difficult to determine since methylxanthines do have other with cholera toxin, prostaglandins, isoproterenol, or epineph- biochemical effects. rine are not feasible for their cost, for their possible side effects, Based on the facts that several discrepancies were observed and because the reciprocal effects of /8-adrenergic agonists between the degree of tumor promoter-increased cyclic nu and TPA on /8-adrenergic receptors (18) and normal adrenergic cleotide levels and that of ODC induction in vivo and that responsiveness of the epidermis (21, 31, 36, 55) have not phosphodiesterase inhibitor treatments raised cyclic nucleotide been clarified. In contrast, IBMX and related phosphodiester- levels as much as did TPA without eliciting a comparable ase inhibitors have consistently been reported to increase ODC induction of ODC activity, it would be tempting to conclude that activity (1, 9, 10, 12, 23, 41, 48) and to enhance its induction the early and transient elevation of epidermal cyclic GMP and by TPA (41). Topical applications of phosphodiesterase inhib cyclic AMP by TPA is unlikely to mediate the mechanism by itors dissolved in acetone have been demonstrated to reach which TPA induces ODC in mouse skin. However, the better the target epidermal cells (60). We chose this mode of appli correlations observed between the production of cyclic nucleo cation not only because it is consistent with the way TPA is tides and induction of ODC activity following incubation of administered to the skin but also because this is certainly a isolated epidermal cells with TPA, IBMX, and cholera toxin more effective way than by systemic routes to act directly have led us to a rather opposite conclusion in a previous report without delay or systemic toxicity on a large number of epider (41). Our various studies illustrate the difficulties inherent in mal target cells. As shown in our results, IBMX or other phos correlating quantitative changes in cyclic nucleotide levels with phodiesterase inhibitors alone raised epidermal cyclic GMP subsequent ODC induction in cells stimulated by a variety of and cyclic AMP to levels corresponding to the effect of TPA agents. It has been emphasized that a stimulation of cyclic but failed to reproduce the induction of ODC activity by TPA. AMP-dependent protein kinase need not be preceded by a Since the ability of IBMX to inhibit, or at least slow down, the proportionately large increase in cyclic AMP (47). Therefore, degradation of cyclic nucleotides appeared to last for about 1 only the study of the activation of cyclic AMP-dependent pro hr, this compound was applied immediately before TPA treat tein kinase, which is a more reliable index or a cyclic AMP- ment in order to alter as much as possible the effects of TPA. mediated event (47), would provide definitive answers con The magnitude of the cyclic nucleotide response in the pres cerning cyclic nucleotide involvement in ODC induction by ence of IBMX was also dependent upon the dose of TPA and TPA. However, the other findings of the present and accom correlated with the promoting abilities of diverse tumor pro panying paper (43) provide new and substantial evidence that, moters. Much higher levels of cyclic GMP and cyclic AMP were under certain conditions, there is a dissociation of Â¡n-

3924 CANCER RESEARCH VOL. 41

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1981 American Association for Cancer Research. TPA, IBMX, Cyclic Nucleotides, and ODC Induction creases in levels of cyclic nucleotides from induction of ODC promotion. In: H. H. Hiatt, J. D. Watson, and J. A. Winsten (eds.), Origins of Human Cancer: Proceedings of the Cold Spring Harbor Conference on Cell activity and skin tumors by the tumor promoter (29). Topical Proliferation, Book B, pp. 773-783. Cold Spring Harbor. N. Y.: Cold Spring applications of cyclic GMP and cyclic AMP in solution in Harbor Laboratory. 1977. acetone were unable, in the presence or absence of IBMX, to 7. Boutwell, R. K. Biochemical mechanisms of tumor promotion. In: T. J. Slaga. A. Sivak, and R. K. Boutwell (eds.), Carcinogenesis, a Comprehensive successfully mimic the stimulatory effect of TPA on ODC. The Survey, Vol. 2, pp. 49-58. New York: Raven Press, 1978. additional inhibition of basal and TPA-induced ODC activities 8. Burton, K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetrie estimation of deoxyribonucleic acid. Biochem. observed after cyclic GMP plus cyclic AMP treatment indicates J., 62. 315-323, 1956. that the exogenously added cyclic nucleotides do enter into 9. Byus, C. V., and Russell, D. H. Effects of methyl xanthine derivatives on the epidermal cells and suggests that both cyclic GMP and cyclic AMP levels and ornithine decarboxylase activity of rat tissues. Life Sci., 15: 1991-1997, 1974. cyclic AMP may actually decrease polyamine biosynthesis. The 10. Byus, C. V., and Russell, D. H. Ornithine decarboxylase activity: control by negative effect of exogenously added cyclic nucleotides on cyclic nucleotides. Science (Wash. D. C.). J87. 650-652, 1975. ODC activity is apparently not due to increased cyclic nucleo- 11. Combest. W. L., and Russell, D. H. Separate mechanisms of induction for ornithine decarboxylase by triiodothyronine and aminophylline. Life Sci., 27. tide phosphodiesterase activities, since this inhibition persists 651-658, 1980. in the presence of IBMX. Cyclic AMP was slightly more effective 12. Costa, M. Cyclic AMP-dependent regulation of ornithine decarboxylase than cyclic GMP in decreasing basal and TPA-stimulated ODC activity in Chinese hamster ovary cells maintained with a salts/glucose medium. J. Cyclic Nucleotide Res., 5. 375-387, 1978. activities, but cyclic AMP was applied at a dose of 0.25 jumol 13. Costa, M. Superinduction of ornithine decarboxylase by actinomycin D and whereas only 0.16 jumol of cyclic GMP was administered to the cordycepin. Biochem. Biophys. Res. Commun., 81: 832-840, 1978. 14. Costa. M., Costa, E. R., Manen, C. A., Sipos, l. G., and Russell, D. H. mice. Furthermore, at physiological levels, cyclic AMP is usu Adenosine cyclic 3':5'-monophosphate-dependent protein kinase and orni ally 10 times more concentrated in the epidermis than is cyclic thine decarboxylase involvement in the induction of cytochrome P-450 and GMP, an observation compatible with the fact that the 2 cyclic hepatic hypertrophy. Mol. Pharmacol., 12: 871-878, 1976. nucleotides have different physiological importance. However, 15. Estensen, R. D., Hadden, J. W., Hadden, E. M.. Touraine, F., Touraine, J. L., Haddox, M. K., and Goldberg. N. D. Phorbol myristate acetate: effects of the levels of epidermal cyclic nucleotides have not been mea a tumor promoter on intracellular cyclic GMP in mouse fibroblasts and as a sured following topical applications of the above doses of cyclic mitogen on human lymphocytes. In: B. Clarkson and R. Baserga (eds.), Control of Proliferation in Animal Cells, pp. 627-634. Cold Spring Harbor, AMP and cyclic GMP to the skin. Therefore, one should not N. Y.: Cold Spring Harbor Laboratory, 1974. discard the possibility that excessively high concentrations of 16. Frandsen. E. K., and Krishna, G. A simple ultrasensitive method for the exogenously added cyclic GMP may interact nonspecifically assay of cyclic AMP and cyclic GMP in tissues. Life Sci., 18: 529-542, with components of the cyclic AMP-dependent system (20), 1976. 17. Garte, S. J., and Belman, S. Effects of multiple phorbol myristate acetate resulting in an effect on ODC activity similar to that obtained treatments on cyclic nucleotide levels in mouse epidermis. Biochem. Bio phys. Res. Commun., 84: 489-494. 1978. with cyclic AMP. Despite the better correlations between in 18. Garte. S. J., and Belman, S. Tumor promoter uncouples /?-adrenergic creases in cyclic nucleotide levels and induction of ODC activity receptor from adenyl cyclase in mouse epidermis. Nature (Lond.), 284: described in vitro (41), a dose-dependent inhibition of basal 171-173, 1980. and TPA-induced ODC activities was reproduced with isolated 19. Garte, S. J.. and Belman, S. Diurnal variation in cyclic nucleotide levels in normal and phorbol myristate acetate treated mouse epidermis. J. Invest. epidermal cells during incubation with diverse concentrations Dermatol.. 74:224-225, 1980. 20. Goldberg, N. D., O'Dea, R. F., and Haddox, M. K. Cyclic GMP. Adv. Cyclic of cyclic nucleotides or their dibutyryl derivatives, in both the Nucleotide Res., 3: 155-223, 1973. presence and the absence of IBMX. Finally, the results pre 21. Grimm. W., and Marks, F. Effect of tumor-promoting phorbol esters on the sented in the accompanying paper (43) indicate that both IBMX normal and the isoproterenol-elevated level of adenosine 3':5'-cyclic mono- and cyclic nucleotide treatments inhibit epidermal ODC activity phosphate in mouse epidermis in vivo. Cancer Res., 34: 3128-3134. 1974. 22. GrÃ¶tendorst, G. R., and Schimmel, S. D. Alteration of cyclic nucleotide levels in situ and decrease dramatically TPA-induced polyamine ac in phorbol 12-myristate 13-acetate treated myoblasts. Biochem. Biophys. cumulation, macromolecule synthesis, and formation of skin Res. Commun.. 93: 301-307, 1980. papillomas. Therefore, although early and transient elevations 23. Hogan, B., Shields, R., and Curtis, D. Effect of cyclic nucleotides on the induction of ornithine decarboxylase in BHK cells by serum and insulin. Cell, of cyclic nucleotides may participate in the molecular mecha 2:229-233, 1974. nism by which TPA induces ODC activity, our data also suggest 24. JÃ¤nne,J., Poso, H., and Raina, A. Polyamines in rapid growth and cancer. Biochim. Biophys. Acta, 473: 241-293, 1978. that high levels of cyclic AMP and cyclic GMP maintained by 25. Kudlow, J. E., Rae. P. A., Gutmann, N. S.. Schimmer, B. P., and Burrow, G. diverse means in the epidermis beyond the initial cyclic nucleo N. Regulation of ornithine decarboxylase activity by corticotropin in adre- tide response to TPA may trigger some inhibitory effects on nocortical tumor cell clones: roles of cyclic AMP and cyclic AMP-dependent protein kinase. Proc. Nati. Acad. Sei. U. S. A., 77: 2767-2771, 1980. the polyamine pathway and on the tumor promotion process. 26. Marks, F., and Raab, I. The second messenger system of mouse epidermis; IV. Cyclic AMP and cyclic GMP phosphodiesterase. Biochim. Biophys. Acta, REFERENCES 334: 368-377. 1974. 27. Marrs, J. M., and Voorhees, J. J. A method for bioassay of an epidermal 1. Bachrach, U. Cyclic AMP-mediated induction of ornithine decarboxylase of chalone-like inhibitor. J. Invest. Dermatol., 56: 174-181. 1971. glioma and neuroblastoma cells. Proc. Nati. Acad. Sei. U. S. A., 72 3087- 28. Matsui, I., Otani, S., and Morisawa, S. Induction of ornithine decarboxylase 3091, 1975. by biliverdin in rat liver. Life Sci., 24: 2231-2236, 1979. 2. Bachrach, U., Katz, A., and Hochman. J. Polyamines and protein kinase. I. 29. Mufson, R. A., Astrup, E. G., Simsiman, R. C., and Boutwell, R. K. Dissocia Induction of ornithine decarboxylase and activation of protein kinase in rat tion of increases in levels of 3':5'-cyclic AMP and 3':5'-cyclic GMP from glioma cells. Life Sci., 22. 817-822, 1978. induction of ornithine decarboxylase by the tumor promoter 12-O-tetra- 3. Baird. W. M., Melera, P. W., and Boutwell. R. K. Acrylamide gel electropho- decanoylphorbol-13-acetate in mouse epidermis in vivo. Proc. Nati. Acad. resis studies of the incorporation of cytidine-3H into mouse skin RNA at early Sei. U. S. A., 74: 657-661, 1977. times after treatment with phorbol esters. Cancer Res., 32: 781 -788, 1972. 30. Mufson, R. A., Fischer, S. M., Verma, A. K., Gleason, G. L, Slaga, T. J., and 4. Belman, S., and Troll, W. Phorbol-12-myristate-13-acetate effect on cyclic Boutwell, R. K. Effects of 12-O-tetradecanoylphorbol-13-acetate and mez- adenosine 3':5'-monophosphate levels in mouse skin and inhibition of phor- erein on epidermal ornithine decarboxylase activity, isoproterenol-stimulated bol-myristate-acetate-promoted tumorigenesis by theophylline. Cancer Res levels of cyclic adenosine 3':5'-monophosphate, and induction of mouse 34. 3446-3455, 1974. skin tumors in vivo. Cancer Res., 39: 4791-4795, 1979. 5. Belman, S.. Troll, W., and Garte, S. J. Effect of phorbol myristate acetate on 31. Mufson, R. A., Simsiman, R. C.. and Boutwell, R. K. The effect of the phorbol cyclic nucleotide levels in mouse epidermis. Cancer Res., 38. 2978-2982. ester tumor promoters on the basal and catecholamine-stimulated levels of 1978. cyclic adenosine 3':5'-monophosphate in mouse skin and epidermis in vivo. 6. Boutwell, R. K. The role of the induction of ornithine decarboxylase in tumor Cancer Res., 37: 665-669, 1977.

OCTOBER 1981 3925

32. Mufson, R. A., Simsiman, R. C., and Boutwell, R. K. Increased cyclic 46. Rochette-Egly, C., Chouroulinkov, I., and Castagna, M. Cyclic nucleotide adenosine 3':5'-monophosphate phosphodiesterase activity in the epidermis levels in rat embryo fibroblasts treated with tumor-promoting phorbol dies- of phorbol ester-treated mouse skin and in papillomas. Cancer Res., 39: ters. J. Cyclic Nucleotide Res., 5: 385-395, 1979. 2036-2040, 1979. 47. Russell. D. H., Byus, C. V., and Manen, C. A. Proposed model of major 33. Murad, F., Manganello, V., and Vaughan, M. A simple, sensitive protein- sequential biochemical events of a trophic response. Life Sci., 19: 1297- binding assay for guanosine 3':5'-monophosphate. Proc. Nati. Acad. Sei. U. 1306, 1976. S.A., 68. 736-739, 1971. 48. Scheinman, S. J., Burrow, G. N., and Theoharides, T. C. Stimulation of 34. Murray, A. W., and Froscio, M. Effect of tumor promoters on the activity of ornithine decarboxylase synthesis in the rat thyroid. Life Sci., 21: 1143- cyclic adenosine 3':5'-monophosphate-dependent and -independent protein 1148, 1977. kinases from mouse epidermis. Cancer Res., 37. 1360-1363, 1977. 49. Slaga, T. J., Fischer, S.U., Nelson, K., and Gleason, G. L. Studies on the 35. Murray, A. W., Solanki, V., Froscio, M., and Rogers, A. Effects of cholera mechanism of skin tumor promotion: evidence for several stages in promo toxin on ornithine decarboxylase activity in mouse skin. J. Invest. Dermatol., tion. Proc. NaÂ«.Acad. Sei. U. S. A.. 77: 3659-3663, 1980. 75. 508-511. 1980. 50. Steiner, A. L., Parker, C. W., and Kipnis, D. M. Radioimmunoassay for cyclic 36. Murray, A. W., Verma, A. K., and Froscio, M. Effect of carcinogens and nucleotides; I. Preparation of antibodies and iodinated cyclic nucleotides. J. tumor promoters on epidermal cyclic adenosine 3':5'-monophosphate me Biol. Chem., 247: 1106-1113, 1972. tabolism. In: W. E. Criss, T. Ono, and J. R. Sabine (eds.). Control Mecha 51. Trosko, J. E., Yager, J. D., Bowden, G. T., and Butcher, F. R. The effects of nisms in Cancer, pp. 217-229. New York: Raven Press, 1976. several croton oil constituents on two types of DNA repair and cyclic 37. O'Brien, T. G. The induction of ornithine decarboxylase as an early, possibly nucleotide levels in mammalian cells m vitro. Chem.-Biol. Interact., 11:191- obligatory, event in mouse skin carcinogenesis. Cancer Res., 36: 2644- 205, 1975. 2653, 1976. 52. Veldhuis, J. D., Harrison, T. S., and Hammond, J. M. /8-2-Adrenergic 38. O'Brien, T. G., Simsiman, R. C., and Boutwell, R. K. Induction of the stimulation of ornithine decarboxylase activity in porcine granulosa cells in polyamine-biosynthetic enzymes in mouse epidermis and their specificity for vitro. Biochim. Biophys. Acta, 627: 123-130, 1980. tumor promotion. Cancer Res., 35. 2426-2433, 1975. 53. Verma, A. K., Froscio, M., and Murray, A. W. Croton oil- and benzo(a)pyrene- 39. Olson, J. W., and Russell, D. H. Prolonged induction of hepatic ornithine induced changes in cyclic adenosine 3':5'-monophosphate and cyclic gua decarboxylase and its relation to cyclic adenosine 3':5'-monophosphate- nosine 3':5'-monophosphate phosphodiesterase activities in mouse epider dependent protein kinase activation after a single administration of diethyl- mis. Cancer Res., 36: 81-87, 1976. nitrosamine. Cancer Res., 39. 3074-3079, 1979. 54. Verma. A. K., Lowe, N. J., and Boutwell, R. K. Induction of mouse epidermal 40. Osterman, J., and Hammond, J. M. Prostaglandin stimulation of ovarian ornithine decarboxylase activity and DNA synthesis by ultraviolet light. ornithine decarboxylase in vitro. Biochem. Biophys. Res. Commun., 63: Cancer Res.. 39: 1035-1040, 1979. 794-799, 1978. 55. Verma, A. K., and Murray, A. W. The effect of benzo(a)pyrene on the basal 41. Perchellet, J. P., and Boutwell, R. K. Enhancement by 3-isobutyl-1-meth- and isoproterenol-stimulated level of cyclic adenosine 3':5'-monophosphate ylxanthine and cholera toxin of 12-O-tetradecanoylphorbol-13-acetate-stim- in mouse epidermis. Cancer Res., 34: 3408-3413, 1974. ulated cyclic nucleotide levels and ornithine decarboxylase activity in iso 56. Verma, A. K., Shapas, B. G., Rice, H. M., and Boutwell, R. K. Correlation of lated epidermal cells. Cancer Res., 40: 2653-2660, 1980. the inhibition by retinoids of tumor promoter-induced mouse epidermal 42. Perchellet, J. P., and Boutwell. R. K. The effects of 3-isobutyl-1-methylxan- ornithine decarboxylase activity and of skin tumor promotion. Cancer Res.. thine (IBMX) on 12-O-tetradecanoylphorbol-13-acetate (TPAHnduced 39:419-425. 1979. cyclic nucleotide levels and ornithine decarboxylase (ODO activity in mouse 57. Vesely, D. L. Biochemical evidence of cocarcinogenesis: tumor promoting epidermis in vivo and in vitro. Proc. Am. Assoc. Cancer Res., 21:121,1980. agent enhances methylnitrosourea activation of rat guanylate cyclase activ 43. Perchellet, J. P., and Boutwell, R. K. Effects of 3-isobutyl-1-methylxanthine ity. Res. Commun. Chem. Pathol. Pharmacol.. 24: 329-338. 1979. and cyclic nucleotides on the biochemical processes linked to skin tumor 58. Voorhees, J. J., Colburn, N. H., Stawiski. M., Duell, E. A., Haddox, M., and promotion by 12-O-tetradecanoylphorbol-13-acetate. Cancer Res., 41: Goldberg, N. D. Imbalanced cyclic AMP and cyclic GMP levels in the rapidly 3927-3935. 1981. dividing, incompletely differentiated epidermis of psoriasis, in- B. Clarkson 44. RÃ¶chelte, C., and Castagna, M. A simultaneous protein-binding assay for and R. Baserga (eds.). Control of Proliferation in Animal Cells, pp. 635-648. adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate in Cold Spring Harbor, N. Y.: Cold Spring Harbor Laboratory, 1974. biological materials. Biochem. Biophys. Res. Commun.. 74: 1287-1296, 59. Voorhees, J. J., Duell, E. A., Stawiski, M., and Harrell, E. R. Cyclic nucleotide 1977. metabolism in normal and proliferating epidermis. Adv. Cyclic Nucleotide 45. Rochette-Egly, C., and Castagna, M. Oscillating levels of adenylate and Res.. 4: 117-162, 1974. guanylate cyclase activities in rat embryo fibroblasts stimulated to divide. 60. Zajdela, F.. and Latarjet, R. Inhibition of skin carcinogenesis in vivo by Biochem. Biophys. Res. Commun., 86. 937-944, 1979. caffeine and other agents. Nati. Cancer Inst. Monogr . 50:133-140, 1978.

3926 CANCER RESEARCH VOL. 41

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1981 American Association for Cancer Research. Effects of 3-Isobutyl-1-methylxanthine and Cyclic Nucleotides on 12-O-Tetradecanoylphorbol-13-acetate-induced Ornithine Decarboxylase Activity in Mouse Epidermis in Vivo

Jean-Pierre Perchellet and R. K. Boutwell

Cancer Res 1981;41:3918-3926.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/41/10/3918

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/41/10/3918. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.