[CANCER RESEARCH 39, 3074-3079, August 1979] 0008-5472/79/0039-0000$02.00 Prolonged Induction of Hepatic Ornithine Decarboxylase and Its Relation to Cyclic Adenosine 3':5'-Monophosphate-dependent Protein Kinase Activation after a Single Administration of Diethylnitrosamine' Jack W. Olson2 and Diane Haddock Russell3 Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, Arizona 85 724 ABSTRACT application of a tumor promoter, 12-O-tetnadecanoylphonbol 13-acetate, a phorbol ester, to mouse skin (38—40).Substantial After a single injection of diethylnitrosamine (200 mg/kg), data have been accumulated in this system to suggest that the there was a rapid increase in the activity ratio of hepatic cyclic induction of onnithine decanboxylase is a specific and essential adenosine 3':S'-monophosphate (cyclic AMP)-dependent pro event for tumor promotion in the 2-stage model for mouse tein kinase (within 1 hr) followed by the induction of ornithine epidenmal cancinogenesis (2). decarboxylase which was detectable by 3 hr. Both the cyclic Kuo and Greengard (32) have postulated that cyclic AMP4 AMP-dependent protein kinase activity ratio and the activity of exerts its influence on cellular metabolism through the activa ornithine decarboxylase were significantly elevated above con tion of cyclic AMP-dependent protein kinase(s). We have re trols for 7 days following the administration of diethylnitnosa ported previously that the induction of onnithine decarboxylase mine. A single noncarcinogenic dose of diethylnitrosamine (25 in several systems seems to be more closely related to the mg/kg) did not increase the cyclic AMP-dependent protein activation of cyclic AMP-dependent protein kinase(s) than to kinase activity ratio on induce onnithine decanboxylase activity the actual intracellular fluctuation of cyclic AMP (4, 8—10). at 24 hr postadministration. However, serial administration of Therefore, an increased activity ratio of cyclic AMP-dependent diethylnitrosamine (25 mg/kg) for 4 or 7 days resulted in an protein kinase probably reflects increased cyclic AMP levels increased activity ratio of cyclic AMP-dependent protein kinase detected by the kinase even though the rapid metabolism of and increased ornithine decanboxylase activity. This is the first cyclic AMP may render alterations in cyclic AMP difficult to report of a prolonged increase in both the activity ratio of measure (5). hepatic cyclic AMP-dependent protein kinase and the activity Considerable evidence indicates that the induction of orni of ornithine decarboxylase in response to a single carcinogenic thine decanboxylase may be mediated through the activation of dose of diethylnitrosamine. cyclic AMP-dependent protein kinase. The rapid activation of cyclic AMP-dependent protein kinase followed by the transcnip INTRODUCTION tional induction of ornithine decarboxylase is consistently ob Polyamines accumulate during cell growth processes and served in response to a tnophic stimulus. This temporal se are generally considered to be the functional organic cations quence of events has been observed following such diverse since their synthesis is sequentially regulated during hypertro trophic stimuli as mitogens (7), drugs that induce enzymes (4, phy and hyperplasia (1, 13, 30, 43, 45). The induction of 15, 36), tnophic hormones (5, 6, 9, 42, 44), analogs of cyclic ornithine decanboxylase, the nate-limiting enzyme in the poly AMP and/on phosphodiesterase inhibitors (8, 11, 17), and amine-biosynthetic pathway, is an early marked event in all compensatory growth systems, such as regenerating rat liver growth systems studied to date (30, 45). Increased onnithine and hypertnophic adrenal gland (6). However, cyclic AMP decanboxylase activity and accumulation of polyamines occur dependent protein kinase activity has not been examined dun during the growth of experimental animal tumors and human ing phonbolester induction of ornithine decarboxylase in mouse tumors (30, 45). skin. Relatively few studies, however, have examined ornithine Alterations in cyclic AMP-dependent protein kinase isozyme decanboxylase activity during the chemical induction of carci activities and/or cyclic AMP-binding proteins have been ob nogenesis. A recent report demonstrated that rats receiving a served in several transformed cell lines (24, 25, 27, 29, 33, complete liven carcinogen (4-dimethylaminoazobenzene) in 34), in rapidly growing hepatomas (1 8), during cell cycle pro their diet had substantially elevated hepatic onnithine decan gression of Chinese hamster ovary cells (16), during embryonic boxylase activity several months prior to the appearance of development (26), and in human mammary and renal carcino visible hepatocellulan carcinomas (48). Dramatic elevations in mas (20, 23). Several studies have examined cyclic AMP onnithine decarboxylase activity occur within a few hr of the dependent protein kinase activity during the chemical induction of carcinogenesis. Lung tumors induced in mice by a single , This work was supported by USPHS Research Grant CA-i 4783 from the i.p. injection of urethan had significantly higher basal and cyclic National Cancer Institute. AMP-stimulated protein kinase activities than did the unin 2 Recipient of Fellowship CA-061 32 from the National Cancer Institute. To whom requests for reprints should be addressed, at Department of Pharmacol volved normal lung tissues (35). Mice fed a diet of 2-acetyl ogy, College of Medicine, Health Sciences Center, The University of Arizona, aminofluorene (500 ppm) for 18 months exhibited significantly Tucson, Ariz. 85724. 3 Recipient of Research Career Development Award CA-00072 from the National Cancer Institute. 4 The abbreviation used is: cyclic AMP, cyclic adenosine 3':5'-monophos Received November 10, 1978; accepted May 8, 1979. phate. 3074 CANCER RESEARCH VOL. 39 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1979 American Association for Cancer Research. Hepatic induction of Ornithine Decarboxylase increased amounts of the cyclic AMP-dependent protein kinase used in the assay was the same as the homogenization buffer. isozymes (46). Ethionine-fed rats had increased hepatic cyclic Ornithine decarboxylase activity was determined as the amount AMP-dependent protein kinase activity by 8 weeks of treatment of 14CO2releasedfrom 0.5 zCiof L-['4C]ornithine at 37°during and, after 38 weeks of ethionine ingestion, cyclic AMP-de a 30-mm assay (47). The assay was stopped with 0.5 ml 1 M pendent protein kinase was activated in the resulting hepato citric acid, and the released ‘4C02wascollected on Whatman mas (19). No. 3MM filter papers prespotted with 20 i1 of NCS (Amen The purpose of this study was to assess the effects of a sham/Searle Corp.). The filter papers were counted in toluene: single carcinogenic dose of a liver carcinogen (diethylnitrosa Omnifluon scintillant. All enzyme activities were corrected for mine, 200 mg/kg) on the time and extent of ornithine decar blanks which contained 4-bromo-3-hydroxybenzyloxyamine boxylase induction and cyclic AMP-dependent protein kinase dihydrogen phosphate in the reaction mixture. The enzyme activation and also on total amounts of type I and type II cyclic activity was linear with respect to incubation time and enzyme AMP-dependent protein kinase isozymes. We report that a concentration. single dose of diethylnitnosamine rapidly activated cyclic AMP Determination of Cyclic AMP-dependent Protein Kinase dependent protein kinase and induced ornithine decarboxyl Activity Ratios. The conditions for tissue preparation and ase. After a single carcinogenic dose of diethylnitrosamine, the assay of cyclic AMP-dependent protein kinase were optimized protein kinase activity ratio and ornithine decarboxylase activity to best preserve the hormonal effects on the enzymes. The remained significantly elevated for at least 7 days, a pattern procedure was similar to that of Cherrington et ai. (12) and not seen after the administration of other growth-producing Byus et ai. (5). Liver samples were rapidly excised and imme stimuli. Although a single noncarcinogenic dose of diethylni diately homogenized using a Bninkman Polytron PT-i 0 homog trosamine did not alter the cyclic AMP-dependent protein ki enizer (full speed, 2 bursts for 5 sec each) in 5 volumes of ice nase activity ratio, serial noncarcinogenic doses resulted in cold 10 mM potassium phosphate buffer, 1 m@EDTA, 5 m@ cumulative increases in the activity ratio of protein kinase and sodium fluoride, 5 mM 2-mercaptoethanol, 0.5 m@3-isobutyl in the ornithine decanboxylase activity. 1-methylxanthine, and 100 m@sodium chloride, pH 6.8. Whole homogenates were centrifuged for 5 mm at 10,000 x g, and MATERIALS AND METHODS the resulting supernatant was diluted with the same buffer to 1: 133 (v/v). Twenty ft of this solution were assayed for protein [-y-32P]ATP (2.0 to 3.5 mCi/mmol) and L-[1-'4C]onnithine (6 kinase activity in the presence and absence of saturating mCi/mmol) were obtained from Amensham/Searle Corp. , An amounts of cyclic AMP (10 @LM)ina total volume of 75 @tl.The lington Heights, Ill. Unlabeled ATP and L-ornithine, cyclic AMP, final incubation mixture contained 20 m@isodium-potassium histone H2b, and Triton X-100 were purchased from Sigma phosphate buffer (pH 6.8), 0.5 mM 3-isobutyl-i -methylxan Chemical Co., St. Louis, Mo. 3-Isobutyl-1 -methylxanthine was thine, 10 mM magnesium chloride, 50 @tgH2bhistone, plus or obtained from Aldrich Chemical Co., Milwaukee, Wis. Diethyl minus 10 @.tMcyclicAMP, 100