[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 phospho- diesterases, 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 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1981 American Association for Cancer Research. TRA, IBMX, Cyclic Nucleotides, and ODC Induction 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 (57° 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 90°. 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.