Prolonged Induction of Hepatic Ornithine Decarboxylase and Its

[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 (38â€”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, 8â€”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

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 37Â°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 â€˜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 @tMATP,and 1 @Ci[â€˜y-32PJATP. nitrosamine was from Eastman Kodak Co., Rochester, N. V. The assay was initiated by the addition of ATP and incubated DEAE-cellulose (DE52) and No. 3MM filter paper were from for 3 mm at 30Â°. Protein kinase activity was determined, Whatman, Clifton, N. J. The silica gel-impregnated glass fiber essentially as described by Huang and Robinson (28), from the sheets (ITLC) were from Gelman Instrument Co., Ann Arbor, amount of 32P incorporated into acid-precipitable material. Mich. Utilizing the above conditions, 32Pincorporation was linear with Male Sprague-Dawley rats (130 to 150 g) received Purina respect to both time and kinase concentration. It was deter laboratory chow and water ad ilbitum and were on a photope mined that the above procedures best preserved the hepatic nod from 7 a.m. to 7 p.m. Diethylnitrosamine dissolved in 0.9% protein kinase activity ratio (â€”cyclic AMP:+ cyclic AMP). NaCI solution (100 mg/mI) was injected i.p. into experimental These optimized conditions help preventthe binding of catalytic groups. Controls received a corresponding volume of 0.9% subunits to the particulate fraction and minimize reassociation NaCI solution. All animals were sacrificed by cervical disloca and dissociation of the subunits of the isozymes (12). The tion between 9 a.m. and I 2 noon, and the livers were rapidly protein kinase activity ratio reflects the amount of total type I removed and chilled. A separate sample of each liven was and type II cyclic AMP-dependent protein kinase activity (+ frozen in liquid nitrogen and stored at â€”80Â°forDEAE-cellulose cyclic AMP) as well as the amount of free catalytic subunit (â€” chromatography. Determinations of protein kinase activity na cyclic AMP) present in the tissue. tios and ornithine decanboxylase activity were performed on Since cyclic AMP-dependent protein kinase is found in both fresh liver samples from each individual animal. All enzyme cytoplasmic and particulate fractions, it is possible that dieth preparations were carried out at 4Â°. ylnitrosamine treatment could alter the amount of cyclic AMP Enzyme Assay for Ornithine Decarboxylase Activity. Liver dependent protein kinase in the particulate fraction. Assess samples were homogenized using a Brinkman Polytron PT-i 0 ment of the amount of cyclic AMP-dependent protein kinase homogenizer (full speed, 2 bursts for 5 sec each) in 5 volumes activity in the particulate fraction is facilitated by solubilization of ice-cold 20 mM sodium-potassium phosphate buffer, 100 with a detergent such as Triton X-i 00 (14). Particulate fractions @zMEDTA, 40 @tMpyridoxal phosphate, 2 mM dithiothreitol, 5 of the initial homogenate from 24-hn-diethylnitrosamine-treated mMsodium fluoride, and 500 @tMphenylmethylsulfonylfluoride, animals were rehomogenized in 0.2% Triton X-i 00 plus original pH 7.2. Homogenates were centrifuged for 10 mm at 48,000 homogenization buffer. There was no difference observed be x g. Fifty j@lof the supernatant fraction were assayed for tween the particulate cyclic AMP-dependent protein kinase ornithine decarboxylase activity in a final volume of 200 @tlin pool in diethylnitrosamine-treated rats and 0.9% NaCI solution 15-mI tapered centrifuge tubes fitted with special rubber stop controls. pers and center wells (Kontes Co., Vineland, N. J.). The buffer DEAE-cellulose Chromatography of Cyclic AMP-depend

AUGUST 1979 3075

ent Protein Kinase Isozymes. Liventissue, which had been profile of free catalytic subunit was determined and was found frozen in liquid nitrogen and stored at â€”80Â°,washomogenized not to overlap with the type I isozyme elution profile (0.01 5 to using a Brinkman Polytnon PT-i 0 homogenizer (full speed, 2 0.035 versus 0.060 to 0.Oi 2 M NaCI, respectively). Controls bursts for 5 sec each) in 5 volumes of ice-cold 5 mM Tnis-HCI for complete association are run on each column separation buffer, 2 mM EDTA, and 3 mM sodium fluoride, pH 7.5. Ho and the flowthrough, wash, and initial gradient fractions were mogenates were centrifuged for 5 mm at 10,000 x g, and the assayed to determine that no free catalytic subunit elutes from resulting supernatant was diluted with the same buffer to i :10 the column. These procedures, along with the estimation of (v/v). Five hundred ,@Iofthis solution were applied to a DEAE kinase recovery, ensure that the isozyme changes detected cellulose column (0.7 x 14 cm) previously equilibrated with are accurate. the same buffer. Protein concentration was assessed by the To assess the recovery rate, the cyclic AMP-dependent method of Bradford (3) to ensure that corresponding amounts protein kinase activity in a 10-id aliquot of the supernatant also of protein were present in each supernatant. Columns were was determined. After chromatography, 75 to 90% of the washed with 14 ml buffer, and the kinases were eluted with a supernatant kinase activity was consistently recovered. The linear gradient of 0 to 0.35 M NaCI in buffer (total volume, 30 determination of recovery includes a factor to compensate for ml). Fractions of 1 ml were collected, and 50-@laliquots were salt inhibition of protein kinase activity in the eluted peaks. This assayed for cyclic AMP-dependent protein kinase activity as inhibition was determined to be 39% for type I and 52% for described. This method of tissue preparation in low-ionic type II by comparing the activity of pooled peak fractions before strength buffer and the slow application of a relatively small and after dialysis to remove the NaCI. Specific activities of the sample size on a proportionately large column under low salt isozymes were calculated for the total kinase activity (pmol/ conditions ensure complete reassociation of the kinase sub mm) eluted in the respective peak (corrected for salt inhibition) units so that total pool sizes of the kinase isozymes can be on the basis of the original supernatant protein applied to the accurately measured by the chromatognaphic separation. This column. methodology prevents runthrough of free catalytic, as occurs in many previously reported studies, which leads to undenesti RESULTS mation of total kinase present, especially the type I isozyme which, without these precautions, tends to stay in the dissoci Alterations in Ornithine Decarboxylase and Cyclic AMP ated state. The type I isozyme peak of kinase activity was dependent Protein Kinase after a Single Administration of shown to represent true reassociated holoenzyme and not to Diethylnitrosamine. Hepatic cyclic AMP-dependent protein ki be contaminated with free catalytic subunit by its dependence nase was activated significantly within i hr after a single i.p. on the addition of exogenous cyclic AMP for maximal activity dose of diethylnitrosamine (200 mg/kg) (Chart i ). The activity (e.g., 0.2 versus 1.0 nmol 32Pper mm pen ml, minus and plus ratio was maximally elevated at 2 hr and returned to control cyclic AMP, respectively). As further assurance, the elution level by 3 hr. Significant activation of cyclic AMP-dependent 0.7 @0 0 0.6

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5 6 HOURSAFTER DIETHYLNITROSAMINEi\DMINISTR@TION Chart 1. Changes in the hepatic activity of ornithine decarboxylase and activation of cyclic AMP-dependent protein kinase(s) following a single carcinogenic dose @ of diethylnitresamine (200 mg/kg p.). Rats were sacrificed at various times after injection, and the enzymes were assayed as described in Materials and Methods. The values obtained from animals given injections of 0.9% NaCIsolution are shown as zero time and did not change throughout the experiment. Each point, represents the mean of triplicate determinations of at least 3 rats. Bars, SE.

3076 CANCERRESEARCHVOL. 39

protein kinase preceded an increase in onnithine decanboxyl of samples from the same liver in buffer containing either 0 on ase activity which was detectable by 3 hr after administration 500 mM sodium chloride, extensive dialysis, and then appli of the carcinogen. By 24 hr, onnithine decanboxylase activity cation to DEAE-cellulose columns. Again, there were no differ was elevated 13-fold and remained significantly elevated for ences in the isozyme profiles of diethylnitrosamine and 0.9% 1i days (Table 1). An apparent biphasic activation pattern was NaCI solution-treated animals in the zero salt or 500 mM salt observed for cyclic AMP-dependent protein kinase, since the extracted samples. activity ratio was elevated again by 14 hr and remained ele vated for 7 days following the administration of diethylnitrosa DISCUSSION mine. Effect of Varying the Amount of DiethylnitrosamineAdmin A dramatic and prolonged increase in both the cyclic AMP istered on Ornithine Decarboxylase Activity and Cyclic AMP dependent protein kinase activity ratio and the induction of dependent Protein Kinase Activity Ratio. The effect of dieth onnithine decarboxylase in rat liven occurred after a single ylnitrosamine upon hepatic ornithine decanboxylase activity carcinogenic dose of diethylnitrosamine (200 mg/kg). Single and cyclic AMP-dependent protein kinase activity ratios was noncarcinogenic doses of diethylnitrosamine [25 mg/kg (41, further examined in rats given 4, 25, 100, 200, and 500 mg of 49)] did not change cyclic AMP-dependent protein kinase or diethylnitrosamine pen kg 24 hr prior to sacrifice (Table 2). onnithine decanboxylase activities when measured 24 hr after Onnithine decanboxylase and cyclic AMP-dependent protein administration of the drug. However, daily treatment with non kinase were increased at a dose of 100 mg/kg with a maximal carcinogenic doses of diethylnitnosamine for 4 or 7 days ne i 3-fold stimulation of ornithine decarboxylase and a 2.5-fold suIted in the activation of cyclic AMP-dependent protein kinase stimulation of cyclic AMP-dependent protein kinase after a and the induction of ornithine decarboxylase (Table 3). Seven dose of 200 mg/kg. A dose of 500 mg/kg produced a smaller days of diethylnitnosamine treatment produced a greaten in increase in both activities, 2.5- and 2-fold, respectively. crease in the magnitude of ornithine decanboxylase induction Alterations in Ornithine Decarboxylase and Cyclic AMP than did 4 days of treatment. This result may be comparable to dependent Protein Kinase after Multiple Administrations of the mouse skin response in which the repeated application of Diethylnitrosamine. Onnithine decarboxylase activity and cyclic AMP-dependent protein kinase activity ratios were meas uned after serial doses of diethylnitrosamine (25 mg/kg i.p.) Table2 for 4 and 7 days (Table 3). Both treatment schedules signifi Effect of varying the amount of diethylnitrosamine administered to cantly elevated ornithine decanboxylase activity and cyclic rats on hepatic ornithine decarboxylase activity and cyclic AMP dependent protein kinase activity ratios AMP-dependent protein kinase activity ratios, with the 7-day Rats were sacrificed 24 hr after i.p. injection of diethylnitrosamine treatment producing a larger increase in activities as assessed or 0.9% NaCI solution, and the enzymes were assayed as described in 24 hr after the last administration. .â€˜MaterialsandMethods.â€• Type I and Type II Cyclic AMP-dependent Protein Kinase decarboxylase ac AMP-dependent pro Isozyme Profiles in Diethylnitrosamine-treated and Control Diethylnitrosa tivity (pmol/30 mm/mg pro tein kinase activity (â€” Liver. DEAE-cellulose chromatography was utilized to deter mine (mg/kg)OrnithineAMP)0i7.7 tein)Cyclic Cyclic AMP:+ cyclic mine the total amount of hepatic type I and type II cyclic AMP (30)425.0 Â± 1718(31)b0.277 Â±0.005 (3)25 Â± 0.5 (2)0.247 Â±0.022 dependent protein kinase activity. Chart 2 is a representative Â± 4.0 (3) Â±0.Oi 1 (3) elution profile of the isozymes 24 hr after treatment with dieth ioo 38.0 Â± 3.5 (2)c 0.404 Â±0.015 (3)' ylnitnosamine (200 mg/kg) or 0.9% NaCI solution. No signifi (6)c50045.020020.0 229.0 Â±16.0 (1 2)c0.301 0.675 Â±0.060 Â±12.0 (3)C0.593 Â±0.051 (3)C cant changes in the isozyme patterns were observed in the a Mean Â± SE. of triplicate determinations on each rat liver. treated group and 0.9% NaCI solution controls. Complete b Numbers in parentheses, number of animals in each group. @ extraction of both isozymes was ensured by homogenization C Data differ from controls ( p 0.05).

Table 1 Changes in the hepatic activityinthe of ornithine decarboxylase and the activation of cyclic AMP-dependent protein kinase(s) i.p.)Rats rat following a single injection of diethylnitrosamine (200 mg/kg @ Thevaluesweresacrificedat thetimesindicated,andtheenzymeswereassayedasdescribedin â€˜MaterialsandMethods.â€˜â€˜ throughouttheobtained from animals given injections of 0.9% NaCI solution are shown as zero time and did not change experiment.Cyclic (pmol/min/Time AMP-dependent protein kinase activity following di- mg protein) ethylnitrosamine Ornithine decarboxylase activity â€”CyclicAMP:+ pretreatmentAMP0 (pmol/30 mm/mg protein) â€”CyclicAMP + Cyclic AMP cyclic 0.2771 17.7 Â± 1718(31)b 269Â± 15(30) 97i Â± 27(30) 4 hr 137 Â±12 (4)C 452 Â± 32 (4)C i 000 Â± 44 (4) O.452' 77(11)21 day 229 Â±16 (12)c 540 Â± 49(11)C 972 Â± days 201 Â±i 7 (5)C 709 Â± 46 (6)C i 062 Â±1i 6 (6) @ 3C7 days i 86 Â±20 (3)C 923 Â±103 (3)C 335 Â± 36 (3)C 0.691 0.382C1days 241 Â±21 (2)C 390 Â± 21 (3)C iO2i Â± 39 (3) 0.29811 days 66 Â±14 (3)C 295 Â± 13 (3) 989 Â± 15 (3) O.368C214 days 20 Â± i .5 (2) 395 Â± 33 (3)C i 073 Â± 55 (3) days 20 Â± 2.0 (2) 239 Â± 16 (3) 841 Â± 30 (3) 0.284

a Mean Â± SE. of triplicate determinations on each rat liver. b Numbers in parentheses, number of animals in each group. @ C Data differ from controls (P 0.05).

AUGUST1979 3077

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1979 American Association for Cancer Research. J. w. Olson and D. H. Russell Table3 Changes in the hepatic activity of ornithine decarboxylase and activation of cyclic AMP-dependent proteini.p.)Rats kinase(s) in the rat following a single or serial injections of diethylnitrosamine (25 mg/kg weresacrificed24 hr after the last daily injectionof diethylnitresamine,andthe enzymeswere @@ assayed as described in Materials and Methods. â€˜â€˜Thevalues obtained from animals given injections of 0.9% NaCIsolutionare shownas zerodailydoses.Cyclic AMP-dependent protein kinase activity No. of daily Ornithine decarboxylase ac- (pmol/30 mm/mg protein) doses of dieth- tivity (pmol/30 mm/mg pro- â€”CyclicAMP: ylnitrosamineAMP0 tein) â€”CyclicAMP + Cyclic AMP + cyclic 17.7Â± 178(31)b 269Â±15(30) 971 Â±27(30) 0.277 1 day 16.4 Â± i .4 (5) 255 Â±13 (5) 894 Â±32 (5) 0.285 4 days 86 Â± 8 (3)C 313 Â±34 (3) 748 Â±36 (3)C 0.41 8@ 7 days 178 Â±20 (3)C 500 Â±14 (3)b 922 Â±22 (3) 0.542'@ a Mean Â± SE. of triplicate determinations of each liver sample. b Numbers in parentheses, number of animals in each group. @ b Data differ from controls (p 0.05).

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Chart 2. Representative elution profiles of 0 DEAE-cellulose chromatography of type I and 2 type II cyclic AMP-dependent protein kinase 0@ in diethylnitrosamine-treated and 0.9% NaCI solution control liver. Rats were sacrificed 24 hr after a single carcinogenic dose of diethyl 02 nitrosamine (200 mg/kg i.p.), and chromatog Zc,) raphy of liver samples was performed as de QE scribed in â€˜Materialsand Methods.â€˜â€˜Sixsep arate columns were run for 3 treated rats and ct 3 0.9% NaCI solution controls. The variation 0@â€”0-.,, of chromatography profiles was less than 10% (numbers in parentheses, percentage of con oc trels). z

25 FRACTION

a phorbol ester tumor promoter increased the magnitude of hepatic tumor nodules (48). In another study, the activation of ornithine decarboxylase induction measured 24 hr after the hepatic cyclic AMP-dependent protein kinase was detected at last administration of the tumor promoter (38â€”40). 8 and 38 weeks after the initiation of dietary ethionine to rats, Cyclic AMP-dependent protein kinase and ornithine decar a diet which produced liven carcinomas in some rats by 38 boxylase activities remained elevated for at least 7 days follow weeks (19). Diethylnitrosamine (200 mg/kg), ethionine, and 4- ing a single carcinogenic dose of diethylnitnosamine in contrast dimethylaminoazobenzene are necnogenic (19, 21, 37, 50), to the transient induction of ornithine decarboxylase observed and the sustained elevation of cyclic AMP-dependent protein following treatment with trophic stimuli (4â€”ii, 17, 36, 42, 44) kinase and onnithine decarboxylase activities produced by onafter the application of a tumor promoter to mouse skin (38â€” diethylnitrosamine could be causally related to selective cellu 40). Several studies have examined either the activation of Ian proliferation of preneoplastic foci as discussed by Farber cyclic AMP-dependent protein kinase on the induction of onni and Solt (22). thine decarboxylase during chemical hepatocancinogenesis. A Therefore, prolonged elevation in hepatic onnithine decan prolonged biphasic activation of nat hepatic ornithine decar boxylase activity may be specific for carcinogenesis and tumor boxylase occurred 1 and 4 months after the onset of chronic formation. Further studies of normal and transformed cells may feeding with 4-dimethylaminoazobenzene, a complete carcin indicate whether the time and extent of onnithinedecarboxylase ogen (48). The second increase in hepatic onnithine decarbox induction is a general requirement for the carcinogenic pnoc ylase activity remained elevated until the onset of detectable ess.

3078 CANCER RESEARCH VOL. 39

ADDENDUM 23. Fossberg, T. M., DÃ¸skeland, S. 0., and Ueland, P. M. Protein kinases in human renal cell carcinoma and renal cortex. Arch. Biochem. Biophys., 189: A recent abstract (31) reports the ability to induce ernithine decarboxylase in 372-381, 1978. a mouse adrenal tumor cell line Yi by the addition of cyclic AMP, cyclic AMP 24. Gharrett, A. J., Malkinson, A. M., and Sheppard, J. A. Cyclic AMP-dependent analogs, or adrenocerticotropic hormone. Several protein kinase mutants, which protein kinases from normal and SV4O-transformed 3T3 cells. Nature they developed, exhibited impaired ability to activate the protein kinase and a (Lend.), 264: 673-675, 1976. consequent attenuation of ornithine decarboxylase induction. The authors sug 25. Granner, D. K. Absence of high affinity adenosine 3',S'-monophosphate gest that cyclic AMP and protein kinase are necessary components in the binding sites from cytesol efthree hepatic-derived cell lines. Arch. Biechem. pathway leading to increased ornithine decarbexylase activity In the mouse Biophys., 165: 359-368, 1974. adrenal tumor cell line Yl and several mutant subclones studied. 26. Haddox, M. K., Reeske, W. R., and Russell, D. H. Independent expression of cardiac type I and II cyclic AMP-dependent protein kinase during murine embryogenesis and postnatal development. Blochim. Biophys. Acts, in REFERENCES press,1979. 27. Hochman, J., Bourne, H. R., Coffine, P., Insel, P. A., Krasny, L., and Melman, 1. Bachrach, U. Function of Naturally Occurring Polyamines. New York: Aca K. L. Subunit interaction in cyclic AMP-dependent protein kinases of mutant demic Press, Inc., 1973. lymphomacells. Proc. NatI. Acad. Sci. U. S. A., 74: 1167â€”1171,1977. 2. Beutwell, R. K. Biochemical mechanism of tumor promotion. In: T. J. Sloga, 28. Huang, K. P. , and Robinson, J. C. A rapid and sensitive assay method for A. Sivak, and R. D. Beutwell(eds.), Carcinogenesis, Vol. 2, pp. 49â€”58.New protein kinase. Anal. Biechem., 72: 593â€”599,1976. York: Raven Press, 1978. 29. lnsel, P. A., Beurne, H. R., Ceffine, P., and Temklns, G. M. Cyclic AMP 3. Bradford, M. M. A rapid and sensitive method for the quantitation of micro dependent protein kinase: pivotal role in regulation of enzyme induction and gram quantities of protein utilizing the principles of protein-dye binding. growth. Science, 190: 896-899, 1975. Anal. Biochem., 72: 248-254, 1976. 30. JÃ nne,J., PÃ¶sÃ¶,H.,and Raina, A. Polyamines In rapid growth and cancer. 4. Byus, C. V.. Costa, M., Sipes, I. G., Brodie, B. B., and Russell, D. H. Biochim. Biephys. Acta, 4 73: 241â€”293,1978. Activation of 3':5'-cyclic AMP-dependent protein kinase and induction of 31. Kudlow, J. E., Rae, P. A., Schimmer, B. P., and Burrow, G. N. Hormonal ernithine decarboxylase as early events in the induction of mixed-function Regulation of Ornithine Decarboxylase in Adrenal Cell Clones by cAMP and exygenases. Proc. Nat. Acad. Sd. U. S. A., 73: 1241 â€”1245,1976. cAMP Dependent Protein Kinase. Clin. Res. , 26: 844A, 1978. 5. Byus, C. V., Haddox, M. K., and Russell, D. H. Activation of cyclic AMP 32. Kuo, J. F., and Greengard, P. Cyclic nucleetide-dependent protein kinases. dependent protein kinase(s) by growth hormone in the liver and adrenal IV. Widespread occurrence of adenesine 3',S'-menephesphate-dependent gland of the rat. J. Cyclic Nucleetide Res., 4: 45â€”54,1978. protein kinase In various tissues and phyla of the animal kingdom. Proc. 6. Byus, C. V., Hedge, G. A., and Russell, D. H. The involvement of cyclic AMP NatI. Acad. Sci. U. S. A., 64: 1349â€”1355,1969. dependent protein kinase(s) in the Induction of ornithine decarbexylase In 33. Lasser, M., and Daniel, V. Altered regulation of cyclic AMP-dependent the regenerating rat liver and in the adrenal gland after unilateral adrenal protein kinase in a mouse lymphoma cell line. Blochim. Blephys. Acta, 482: ectemy. Biochim. Biephys. Acta, 498: 39-45, 1977. 41â€”51,1977. 7. Byus, C. V., Klimpel, G. R., Lucas, D. 0., and Russell, D. H. Ornithine 34. Mackenzie, C. W., and Stellwagen, R. H. Differences between liver and decarboxylase induction in mitegen-stimulated lymphocytes is related to the hepatema cells in their complements of adenosine 3':S'-monephesphate specific activation of type I adenesine cyclic 3',S'-monephesphate-depend binding proteins and protein kinases. J. Biol. Chem., 249: 5755â€”5762, ent protein kinase. Mel. Pharmacol., 14: 431 â€”441,1978. 1974. 8. Byus, C. V., and Russell, D. H. Effects of methylxanthine derivatives on 35. Malkinsen, A. M., Gunderson, T. J., and McSwigen, C. E. Protein phosphe cyclic AMP levels and ornithine decarbexylase activity of rat tissues. Life rylation in normal and neeplastic development. Blochem. J., 168: 319â€”321, Sci., 15: 1991-1997, 1974. 1977. 9. Byus, C. V., and Russell, D. H. Ornithine decarbexylase activity: control by 36. Manen, C. A., Costa, M., Sipes, I. 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AUGUST1979 3079

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1979 American Association for Cancer Research. 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. Olson and Diane Haddock Russell

Cancer Res 1979;39:3074-3079.

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