Studies on Adrenocortical Carcinoma of Rat Cyclic Nucleotide Phosphodiesterase Activities'

[CANCER RESEARCH 32, 1734â€”1736,August 1972]

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' ,5â€˜-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' ,5â€˜-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 â€˜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 4Â°.This enzymic preparation was stored at â€”20Â° 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 30Â°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

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 @flÂ°. rates of hydrolysis of 6 of the cyclic nucleotides at a relatively SubstrateNormaltumorcAMP146Â±3Â°30Â±1cGMP160Â±228Â±1cIMP@'145Â±723Â±1cUMP25Â±110Â±1cCMP15Â±13Â±1cTMP33Â±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 16Â± 1 Enzyme kinetic constants of cAMP phosphodiesterase Km and Vmax were determined by nonlinear regression methods as a Mean Â± S.E. described in â€œMaterials and Methods.â€• 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 Â±3.119.93 Â± terminated by the addition of 0.05 ml of 55% chilled rat tumor17.8 Â±1.84.15 Â±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 30Â°. The enzyme kinetic constants, Km and Vmax â€˜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 â€˜ â€˜ , , 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 â€œhyperâ€•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

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' ,5â€˜-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. J. Biol. phosphodiesterase activity (1 , 8, 16) have demonstrated the Chem.,237:1244â€”1250,1962. presence of multiple cyclic phosphodiesterase enzyme system 3. Cheung, W. Y. Properties of Cyclic 3',5'-Nucleotide in most of the tissues analyzed. The multiplicity of this Phosphodiesterase from Rat Brain. Biochemistry, 6: 1079â€”1087, enzyme system was substantiated when the supernatants of 1967. the homogenates of various tissues of the rabbit and rat were 4. Cleland, W. W. Computer Programmes for Processing Enzyme examined by starch-gel electrophoresis (10). However, as Kinetic Data. Nature, 198: 463â€”465,1963. shown in Chart 1, A and B, the normal adrenal and 5. Fiske, C. H., and SubbaRow, Y. The Colorimetric Determination of Phosphorous. J. Biol. Chem., 66: 375 â€”400, 1925. adrenocortical carcinoma tissues of the rat show that there is 6. Kitabchi, A. E., and Sharma, R. K. Corticosteroidogenesis in no break in the slope of the curve, thus suggesting 1 Km for Isolated Adrenal Cells of Rats. I. Effect of Corticotropins and cAMP phosphodiesterase enzyme. These results are of interest, 3',5'-Cyclic Nucleotides on Corticosterone Production. since the concept of high and low Km enzymes for cAMP has Endocrinology,88:1109â€”1116,1971. been proposed (16) to explain the regulation of intracellular 7. Kitabchi, A. E., Wilson, D. B., and Sharma, R. K. Steroidogenesis concentrations of cAMP. The low Km cyclic in Isolated Adrenal Cells of Rat. II. Effect of Caffeine on ACTH phosphodiesterase enzyme, which would be membrane bound and Cyclic Nucleotide-Induced Steroidogenesis and Its Relation to in such a case, in conjunction with adenyl cyclase, could Cyclic Nucleotide Phosphodiesterase (PDE). Biochem. Biophys. contribute to control of the availability of cAMP necessary Res.Commun.,44: 898â€”904,1971. for the regulation of various metabolic functions of the 8. Loten, E. G., and Sneyd, J. G. T. An Effect of Insulin on Adipose Tissue Adenosine 3',S'-Cycic Monophosphate Phosphodiesterase. hormones. Of particular interest are the very low Biochem. J., 120: 1.87â€”193,1970. phosphodiesterase activities toward all 6 of the cyclic 9. Lowry, 0. H., and Lopez, J. A. The Determination of Inorganic nucleotides tested in rat adrenal tumor as compared to the Phosphate in the Presence of Labile Phosphate Esters. J. Biol. normal rat. Since the Km values of the enzymes in both tissues Chem., 162: 421â€”428,1946. are the same, the implication is that there is considerably 10. Monn, E., and Christiansen, R. 0. Adenosine-3, 5'-Monophosphate lowered enzymic activity in the adrenal tumor, possibly Phosphodiesterase; Multiple Molecular Forms. Science, 1 73: because of a lower amount of enzyme produced. Nevertheless, 540â€”542, 1971. that the rat adrenocortical carcinoma 494 has developed an 11. Ney, R. L., Hochella, N. J., Grahme-Smith, D. G., Dexter, R. N., independent method for maintaining high cellular cAMP levels and Butcher, R. W. Abnormal Regulation of Adenosine is an attractive possibility. This possibility seems substantiated, 3',S'-Monophosphate and Corticosterone Formation in an since cAMP levels of the rat adrenal tumor are reported to be Adrenocortical Carcinoma. J. Cliii. Invest., 48: 1733â€”1739, 1969. 12. Sayers, G., Swallow, R. L., and Giordano, N. D. An Improved relatively high and are unaffected by further stimulation of the Technique for the Preparation of Isolated Rat Adrenal Cells. A adenyl cyclase by ACTH (1 1). Sensitive, Accurate and Specific Method for the Assay of ACTH. These studies further support our previously proposed Endocrinology,88: 1063â€”1068,1971. hypothesis that the steps of steroidogenesis in the adrenal 13. Schorr, I., and Ney, R. L. Abnormal Hormone Responses of an tumor are impaired after the adenyl cyclase step (14). From Adrenocortical Cancer Adenyl Cyclase. J. Chin. Invest., 50: the present studies, the above hypothesis may be extended to 1295â€”1300,1971. suggest that the lack of corticosteroidogenic response to 14. Sharma, R. K., and Hashimoto, K. Ultrastructural Studies and ACTH in the tumor may be at a site beyond the formation of Metabolic Regulation of Isolated Adrenocortical Carcinoma Cells cAMP. of Rat. Cancer Res., 32: 666â€”674,1972. 15. Snell, K. C., and Stewart, H. L. Variations in Histological Pattern and Functional Effects of a Transplantable Adrenal Cortical Carcinoma in Intact, Hypophysectomized and Newborn Rats. J. Natl.CancerInst.,22:1119â€”1132,1959. 16. Thompson, W. J., and Appelman, M. M. Multiple Cyclic Nucleotide ACKNOWLEDGMENTS Phosphodiesterase Activities from Rat Brain. Biochemistry, 10: 311â€”316,1971. 17. Thompson, W. J., and Appelman, M. M. Characterization of Cyclic The author wishes to express his appreciation to Dr. James Brush Nucleotide Phosphodiesterases of Rat Tissues. J. Biol. Chem., 246: and Dr. Abbas Kitabchi for useful and stimulating discussions. The 3145â€”3150, 1971. excellent technical assistance of Mrs. Margaret Cirtain is also 18. Wilkinson, G. N., Statistical Estimations in Enzyme Kinetics. appreciated. Biochem. J., 80: 324â€”332,1961.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1972 American Association for Cancer Research. Studies on Adrenocortical Carcinoma of Rat Cyclic Nucleotide Phosphodiesterase Activities

Rameshwar K. Sharma

Cancer Res 1972;32:1734-1736.

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