[CANCER RESEARCH 32, 1734—1736,August 1972] Studies on Adrenocortical Carcinoma of Rat Cyclic Nucleotide Phosphodiesterase Activities' Rameshwar K. Sharma Laboratories of Endocrinology and Metabolism, Veterans Administration Hospital and Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38104 SUMMARY explained on the basis of phosphodiesterase activities of the tissue. Cyclic 3',5'-nucleotide phosphodiesterase activities of the homogenates of normal adrenal and adrenocortical carcinoma tissues of the rat have been measured. The rates of hydrolysis MATERIALS AND METHODS of cyclic adenosine 3' ,5'-monophosphate (cAMP), cyclic guanosine 3' ,5‘-monophosphate, cyclic inosine 3' ,5'-mono Animals. Adrenocortical carcinoma 494, a spontaneously phosphate, cyclic uridine 3',5'-monophosphate, cyclic cytidine occurring tumor discovered in Osborne-Mendel rats by Snell 3' ,5‘-monophosphate, and cyclic thymdine 3' ,5' -monophos and Stewart (1 5), has been maintained in our laboratories by phate by the homogenates of normal rat adrenal were i.p. and s.c. transplantation in Sprague-Dawley rats at 3 to 4 considerably higher than those shown by the homogenates of weeks of age. Of the rats receiving implantation, 60 to 70% the adrenocortical carcinoma of the rat. A multiplicity of successfully developed the tumor. Km ‘5forcAMP phosphodiesterase enzymes was not observed Chemicals. cAMP-3 H was purchased from New England in either the normal rat adrenal or in the adrenocortical tumor. Nuclear, Boston, Mass. Nonradioactive cyclic nucleotides and The Km values for cAMP phosphocliesterases of normal Crotalus atrox snake venom were purchased from the Sigma adrenal and adrenocortical carcinoma 494 were 23.9 and Chemical Company, St. Louis, Mo., and Boehringer 17 .8 j.iM, respectively. These results suggest that lack of Mannheim, New York, N.Y. All other chemicals were corticosteroidogenic response to adrenocorticotropin in the purchased commercially and were of analytical reagent grade. tumor may not be explained on the basis of its Cyclic 3',5'-Nucleotide Phosphodiesterase. Cyclic phosphodiesterase activity but may be at a site beyond the 3',S'-nucleotide phosphodiesterase was obtained from the rat formation of cAMP. adrenal tumor tissue and normal rat adrenal by the method of Cheung (3). The tissues were homogenized in 5 volumes of ice-cold, glass-distilled water, sonically dispersed, and then INTRODUCTION centrifuged at 30,000 X g for 30 ruin. The supernatant solution was then dialyzed against 20 mM Tris-HC1, pH 7.5, We have recently reported an isolated adrenocortical for 48 hr at 4°.This enzymic preparation was stored at —20° carcinoma 494 cell preparation that is useful for study of the in 1-nil aliquots. Once the aliquots were thawed for assay, the abnormalities in control mechanisms of steroidogenesis in remainder of the enzyme preparation was rejected. isolated target tissue (14). In contrast to the isolated cells Assay of Cyclic 3',S'-Nucleotide Phosphodiesterase prepared from the adrenal gland (6, 12), it was demonstrated Activities. Rate of hydrolysis of various cyclic nucleotides was that the tumor cells are not stimulated for corticosteroidogen measured by the method of Butcher and Sutherland (2) as esis by ACTH,2 cAMP, N@-2'O-dibutyryladenosine 3',5'-mono modified by Cheung (3), and the Km values of the phosphate, cyclic inosine 3',5'-monophosphate, or cGMP (14). phosphodiesterase preparations for cAMP were evaluated by This is in spite of the fact that the adenyl cyclase activity in the modified radioactive method of Loten and Sneyd (8), response to ACTH is higher in the tumor than in the normal substituting QAE Sephadex (Cl @)for Bio-Rad AC1-X2 C1 adrenal (1 3). For a better understanding of the control colunm in the procedure for the isolation of adenosine. mechanisms of ACTH or cAMP for the lack of corticosteroido The assay mixture for phosphodiesterase activities consisted genesis, the phosphodiesterase activities of the adrenals of of 40 mM Tris-HC1 (pH 7.5), 1.8 mM MgSO4 , 2 mM cNMP, normal rat and adrenocortical carcinoma of the rat were and crude cyclic phosphodiesterase in a total volume of 0.5 determined. These studies suggest that the lack of ml. steroidogenesis to ACTH or cAMP in the tumor cells cannot be The reaction was started by the addition of cNMP. Incubation was carried out at 30°for 60 mm. After 50 mm, I This work was supported by Veterans Administration Institutional 0.5 mg of Crotalus atrox snake venom in 0.05 ml of 10 mM grants. Tris-HC1 (pH 7.5) was added to convert the 5'-nucleotide 2 The abbreviations used are: ACTH, adrenocorticotropin; cAMP, cyclic adenosine 3',S'-monophosphate; cGMP, cyclic guanosine produced to nucleoside, and the reaction mixture was 3',S'-monophosphate; cNMP, cyclic mononucleotide. incubated further for 10 mm. For every nucleotide, a control Received February 8, 1972; accepted May 1, 1972. tube without phosphodiesterase was included to correct for 1734 CANCER RESEARCH VOL.32 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1972 American Association for Cancer Research. Cyclic Nucleotide Phosphodiesterase Activities Table1 normal and cancer tissue in the rat. The cyclic Hydrolysis of cyclic nucleotides by adrenal homogenates phosphodiesterase activity of rat adrenal tumor was shown to Incubation mixture was as described in the text. Each analysis was be inhibited in a noncompetitive fashion by cGMP (R. K. done in quadruplicate. Phosphodiesterase activity is expressed as Sharma, unpublished observations). As shown in Table I , the nmoles of P@released per mg protein in 60 mm at @fl°. rates of hydrolysis of 6 of the cyclic nucleotides at a relatively SubstrateNormaltumorcAMP146±3°30±1cGMP160±228±1cIMP@'145±723±1cUMP25±110±1cCMP15±13±1cTMP33±rat adrenalRat adrenal high substrate concentration (2 mM ) have been determined in the different tissues, confirming the earlier findings from our laboratories (7). Normal rat adrenals showed significant cyclic phosphodiesterase activity for 6 of the cyclic nucleotides. Table 2 16± 1 Enzyme kinetic constants of cAMP phosphodiesterase Km and Vmax were determined by nonlinear regression methods as a Mean ± S.E. described in “Materials and Methods.― b cIMP, cyclic inosine 3',5'-monophosphate; cUMP, cyclic uridine 3',5'-monophosphate; cCMP, cyclic cytidine 3',S'-monophosphate; cTMP, cyclic thymidine 3',5'-monophosphate. of adenosine mm)NormalTissueKm (jiM)(pmoles formed/60 trace hydrolysis of cNMP by snake venom. The reaction was 0.51Adrenalrat adrenal23.9 ±3.119.93 ± terminated by the addition of 0.05 ml of 55% chilled rat tumor17.8 ±1.84.15 ±0.08 trichioroacetic acid. The reaction mixtures were centrifuged, and the supernatants were assayed for inorganic phosphate by the method of Fiske and SubbaRow (5) as modified by Butcher and Sutherland (2). Protein was determined by the method of Lowry and Lopez (9), with bovine serum albumin as standard. All assays were performed in quadruplicate. The cyclic phosphodiesterase values (Table 1) are expressed as nmoles of P@released per mg in 60 mm at 30°. The enzyme kinetic constants, Km and Vmax ‘for cAMP V hydrolysis by various cyclic phosphodiesterase preparations were determined by use of an incubation mixture that consisted of 40 mM Tris-HC1 buffer, pH 7.5 , 1.8 mM MgSO4, cAMP-3 H (approximately 20,000 dpm), and varying concentrations of nonradioactive cAMP. The reaction was started by the addition of the enzyme preparation, and the incubation was carried out as described above. The reaction V/LSJX IO@ (jaM@) was stopped by the addition of 50 j.il of 10 mM adenosine in 50 mM EDTA. Adenosine formed during the reaction was B ‘ ‘ , , I removed from the unreacted cAMP by passing the reaction mixture through QAE Sephadex (C1) column and eluting the column (5 x 0.5 cm) with 4 ml of distilled water. Radioactivity in aqueous solutions was determined by 0.05 counting a sample in 15 ml Bray's solution in the Nuclear Chicago Mark II automatic liquid scintillation counter. 0.04 Although not shown, cAMP hydrolysis was directly proportional to enzyme concentration. Enzyme concentration 0.03 used in each tube was calculated to give less than 10% substrate decomposition. cAMP concentrations used to 0.02 determine Km values ranged from 0.00625 to 2 mM. A computer program “hyper―fordetermining enzyme 0.0I kinetics constants was that of Cleland (4). It is based on the nonlinear regression methods described by Wilkinson (18). (pM@') RESULTS AND DISCUSSION Chart 1. A , Hofstee kinetic plot for cAMP phosphodiesterase homogenate preparation from rat adrenal tissue. Enzyme activity was The Km values of cAMP phosphodiesterases are 23.9 and measured as described in the text; B, Hofstee kinetic plot for cAMP 17.8 pM , respectively, for normal adrenal and adrenocortical phosphodiesterase homogenate preparation from rat adrenocortical carcinoma of the rat (Table 2). The results show that there is carcinoma tissue. Enzyme activity was measured as described in the no significant difference in the Km values for cAMP between text. AUGUST 1972 1735 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1972 American Association for Cancer Research. Rameshwar K. Sharma cAMP, cGMP, and cyclic inosine 3',S'-monophosphate were REFERENCES degraded at the highest rates, followed by cyclic thymidine 3' ,5,-monophosphate and cyclic uridine 3' ,5‘-monophosphate. 1. Beavo, J. A., Hardman, J. G., and Sutherland, E. W. Cyclic GMP The nucleotide degraded at the lowest rate was cyclic cytidine and Cyclic AMP Hydrolysis by Phosphodiesterase Preparation from 3',S'-monophosphate. The rat adrenal tumor, however, showed Beef and Rat Tissues. Federation Proc., 29: 2077, 1970. considerably lower phosphodiesterase activities for all the 2. Butcher, R. W., and Sutherland, E. W. Adenosine 3',S'-Phosphate in Biological Materials. I. Purification and Properties of Cyclic cyclic nucleotides, as compared to the normal adrenal. 3',S'-Nucleotide Phosphodiesterase and Use of This Enzyme to The radioactive methods for assay of cyclic Characterize Adenosine 3',5'-Phosphate in Human Urine.