- Email: [email protected]
Adenylate cyclase and protein phosphokinase activities in human thyroid. Comparison of normal glands, hyperfunctional nodules and carcinomas
Europ. J. Cancer Vol. 12, pp. 447-453. Pergamon Press 1976. Printed in Great Britain
Adenylate Cyclase and Protein Phosphokinase Activities in Human Thyroid. Comparison of Normal Glands, Hyperfunctional Nodules and Carcinomas* G. SAND~', A. JORTAY++, R. POCHET~ and J. E. DUMONT~ ~Institut de Recherche Interdisciplinaire, Facultg de Mgdecine, Universitd de BruxeUes and Biology Department§, Euratom, and +Service de Chirurgie de l'Institut Bordet, B-I O00 BruxelIes, Belgium A b s t r a c t - - B a s a l and stimulated adenylate cyclase and protein phosphokinase activities
were measured in normal and diseased human thyroids. Seven specimens of thyroid carcinomas (2 papillary, 2 mixed papillo-follicular or trabecular, 1 follicular and 2 medullary), and 6 hyperfunctional autonomous nodules were obtained at thyroidectomy. Nine specimens of normal tissue, taken from the opposite lobe of the nodular thyroids and from entirely normal glands, constituted the control group. Biochemical observations were the following: adenylate cyclase: basal activity and stimulation by T S H were rather similar in all cases. Hyperfunctional nodules were characterized by a significant increase of stimulation, as compared to the normal, by sodium fluoride (13'0 vs 7"S fold) and prostaglandin E x (2'6 vs l'3 fold). Adrenalin had no significant effect. Protein phosphokinase: basal activity was significantly higher in carcinomas by a factor of up to 4-fold. Activation by cyclic A M P was similar in normal and pathological homogenates. These data show that adenylate cyclase activity is not unequivocally related to the degree of malignancy in human thyroid tumors and that protein phosphokinase activity is increased in such tissues. The data are discussed within the framework of current hypotheses derived from studies of transformed cells in vitro.
INTRODUCTION
resulting in a decrease of cyclic A M P concentrations in transformed cells [1]. However, in apparent contradiction with this observation, cyclic A M P levels in rapidly growing animal tumors are high [1] and tropic hormones, such as thyrotropin (TSH), raise cyclic A M P levels and activate growth in their target tissues [3]. The aim of the present work was to compare the activity of two key enzymes of the cyclic A M P system: adenylate cyclase and cyclic AMP-dependent protein phosphokinase in normal and cancerous h u m a n thyroid tissue, and in autonomous hyperfunctioning nodules.
TH~ CYCLmA M P (adenosine 3',5'-cyclic monophosphate) system has been shown to be one of the major intracellular systems for relaying a hormone message from the plasma membrane to the nucleus. It has been implicated in cell differentiation and growth in in vitro preparations [1]. In fibroblasts in culture there is an inverse relation between growth and intracellular levels of cyclic A M P [2]. Moreover an essential event in transformation appears to be a depression in adenylate cyclase activity Accepted 15 January 1976. *This work was supported by a grant from the "Fonds Cancdrologique de la Caisse Gdndrale d'Epargne et de Retraite", Belgium. §Publication No. BIO 1200.
MATERIAL A N D M E T H O D S
Seven specimens of thyroid carcinomas were obtained from patients at thyroidectomy. There 447
448
G. Sand, A. Jorlay, R. Pochet and dr. E. Dumont
were 2 p a p i l l a r y , 2 m i x e d papillo-follicular or trabecular, 1 follicular a n d 2 m e d u l l a r y c a r c i n o m a s . T h e patients r a n g e d in age f r o m 7 to 66; 5 w e r e f e m a l e a n d 2 w e r e m a l e . N o n e o f these p a t i e n t s h a d received previous neck i r r a d i a t i o n . 131I iodide u p t a k e s w e r e w i t h i n n o r m a l limits in all p a t i e n t s a n d t h y r o i d scans u n i f o r m l y d e m o n s t r a t e d low or absence o f activity o v e r t u m o r sites. Six h y p e r f u n c t i o n a l nodules c h a r a c t e r i z e d b y elevated 131I iodide u p t a k e ( 5 0 % at the 6 t h h r ) a n d m a r k e d h y p e r a c t i v i t y at scan were excised. I n 3 cases, the T a suppression test a n d T S H s t i m u l a t i o n o f the s u r r o u n d i n g tissue c o n f i r m e d the diagnosis (see T a b l e 2) [4, 5]. All p a t i e n t s were f e m a l e a n d r a n g e d in age f r o m 33 to 40. N i n e specimens o f n o r m a l tissue t a k e n f r o m the opposite lobe o f the n o d u l a r thyroids a n d f r o m entirely n o r m a l glands constituted the control group. F o r n o r m a l thyroids a n d h y p e r f u n c t i o n a l
nodules, the v a s c u l a r s u p p l y was i n t e r r u p t e d 3 m i n or less before the s p e c i m e n was excised. T h i s t i m e i n t e r v a l was c o n s i d e r a b l y longer for c a r c i n o m a s a n d r a n g e d f r o m 10 to 30 min. T h e excised tissue was i m m e d i a t e l y p l a c e d in n o r m a l saline solution at 0°C, t r a n s p o r t e d to the l a b o r a t o r y , a n d frozen in liquid n i t r o g e n until the t i m e o f b i o c h e m i c a l m e a s u r e m e n t s . I n 1 e x p e r i m e n t with n o r m a l thyroid, the tissue was cut in 6 samples, 3 were assayed freshly a n d the 3 others w e r e frozen. Before freezing, a p o r t i o n was t a k e n for histologic e x a m i n a t i o n in s t a n d a r d h e m a t o x y l i n a n d eosin sections; 15 different fields d i s t r i b u t e d in the 3 m o s t r e p r e s e n t a t i v e sections o f each s p e c i m e n were observed. T h e s t r u c t u r a l p a t t e r n o f t h y r o i d tissue was defined as either h o m o g e n e o u s or heterogeneous after c o m p a r i s o n o f nodules a n d c a r c i n o m a s with n o r m a l thyroids. Criteria used were the r e g u l a r i t y o f follicle size, presence o f colloid cysts, a m o u n t o f c o n n e c t i v e tissue a n d
Table 1. Histologic and biochemical results obtained on thyroid carcinomas. DNA and protein content are expressed in mg/g wet tissue, adenylate cyclase activity in picomoles cyclic A M P formed/lO min/#g DNA, and protein phosphokinase activity in picomoles of phosphate incorporated into protein/min/mg DNA. Effectors added: TSH: 10 mU/ml ; sodium fluoride ( NaF) : 1"10 - 2 M ; adrenalin: 5"10 - 4 M ; prostaglandin E a : 5pg/ml ; cyclic A M P : 2"10 . 6 M Adenylate cyclase Carcinoma
Structural aspect
Cells Colloid
M.V. M.S. W.S. A.R. G.S.
Papillary Papillary Mixed Mixed Follicular
Homog. Homog. Homog. Homog. Heterog.
High Variable High High High
2.8 1.6 0.9 3.5 2-7
134 71 68 152 172
8.9 2.4 1.9 1.4 --
J.M. S.V.
Medullary Heterog. Medullary Heterog.
Low Variable
0.6 1.6
40 64
1.0 .
Patient
Pro+ DNA teins Basal TSH
.
+ NaF
11.0 4.5 2.1 2"8 1.6
60.0 16.7 -12.5 22.5
1"2
5.8 .
.
Protein phospho+ + kinase Adren. PGE I Basal +cAMP 14.6 2.1 -1.7 2.5
-4.0 -3.3 5.0
916 446 934 481 2,120
1.1
1.2
1,015 1,355 1 , 2 5 0 1,510
.
1,125 1,325 1,540 494 3,750
Table 2. Clinical definition and histological results of hyperfunctional nodules. PB 1271 is expressed in pg °/oo, 1 3xi uptake in ~ of tracer dose and PB 131I in mg/L plasma. T3 suppression: 0 = no suppression; - - not done
131I Uptake 6th hr 24th hr
TSH activation of T3 Sup- surrounding Pathological PB131I pression tissue findings
Patient
pB127I
J.C. M.F. J.W.
5.5 I 1.7 5" 1
28.9 63.8 53.9
42.5 60-3 78.8
0.30 0-35 0.49
--0
--+
C.E.
9-5
--
70.0
0"48
--
--
M.I. M.D.
6.5 7.7
--69.6
69.0 76.5
0" 14 1.54
-0
+ +
Structural aspect
\ Cylindrical Heterog. J epithelium Heterog. ] Colloid Homog. Goiter Homog. (Flattened epithelium) Homog. Colloid Heterog. cysts
Population cells~colloid Variable Low Average Low Low Variable
Adenylate Cyclase and Protein Phosphokinase Activities in Human Thyroid fibrosis or degenerative changes disrupting the basic follicular organization. Cell density was roughly evaluated using similar criteria applied to normal and pathological tissues. The cell population was expressed as low or average (typical of normal thyroid) or as high when colloid material was almost totally replaced by cell proliferation (mainly in carcinomas), or as variable if cell population varied greatly from one area to another. From 3 days to 2 months after excision, the frozen glands were thawed, rapidly trimmed free of fat and surrounding connective tissue, sliced with a Stadie Riggs microtome, chopped with a MacIlwain slicer, and homogenized with a motor driven teflon glass homogenizer by 4 strokes at a low speed in ice-cold medium (0"25M sucrose, 0"05M tris-HC1 buffer, pH 7"4). The preparation was filtered through a teflon sieve and used for enzymatic assays (homogenate). Protein content was assayed by the method of Lowry et al. [6] with bovine serum albumin as standard, and DNA by the method of Burton [7]. Adenylate cyclase was measured as previously described [8]. Briefly, aliquots of the homogenate were incubated 10 rain at 30°C in a medium (200/~1) containing 50 m M tris-HC1 buffer, pH 7"4, 2 m M cyclic AMP, 5 m M MgC12, 3 m M ATP, 5/~Ci a32p-ATP (Radiochemical Centre, Amersham, U.K.), 0"1% cristalline bovine serum albumin, 1 0 m M creatine phosphate and 60 pg cristalline rabbit muscle creatine kinase. At the end of the incubation, the test tubes were transferred to an ice bath and 100 pl of an ice-cold solution containing 20 m M cyclic AMP, 50 m M ATP and 3H-cyclic AMP (20,000 counts/rain) were added. Cyclic AMP was then isolated by elution on dry neutral alumina columns [9]. The eluate was diluted in Bray's solution and 3H and a2p radioactivities were counted in a liquid scintillation counter. Protein phosphokinase activity was assayed in a total volume of 1 ml: the complete reaction mixture contained enzyme plus 0"1 M potassium phosphate buffer pH 6"5, 1 m M EDTA, 1 0 m M magnesium acetate, 1 0 m M sodium fluoride, 2 m M theophylline, 2 mg of a mixture of histones (from calf thymus type II, Sigma), 0"2 m M ~-32p-ATP (15-20 dis. min-1/p mole) and, when added, 2 pM cyclic AMP. Assays were carried out at 30°C. After 4 min preincubation, the reaction was initiated by the addition of ~-32p-ATP and stopped after 30 sec, 1 min, 2 min and 4 min by the addition
449
of 1 ml of 40% trichloroacetic acid (TCA). The TCA precipitable material was washed two times by dispersion in cold N a O H 1N and reprecipitation in T C A 200/o. The final TCA precipitate was digested in 1 ml soluene-100 (Packard, Warenville, Ill., U.S.A.) and counted in toluene-omnifluor 4% (NEN, Boston, Mass., U.S.A.) with a Nuclear Chicago Mark II liquid scintillation counter (efficiency of 8090% for 32p). Enzyme activity was linear with time up to 4 m i n and up to a protein concentration of 1 mg. Cyclic AMP, ATP, phosphocreatine, creatine kinase and histones were purchased from Sigma (Saint Louis, Mo., U.S.A.), and adrenalin from Parke-Davis. Other reagents were of analytical grade (Merck, Darmstadt, F.R.D.). Bovine thyrotropin was obtained from Armour and from the N.I.H. Prostaglandin E t w a s a generous gift of Dr. J. Pike, Upjohn Co., Kalamazoo, Michigan, U.S.A. y-32p-ATP was prepared according to the method of Glynn and Chappell [10] modified by Walsh et al. [11], or was obtained from the Radiochemical Centre (Amersham, Great Britain). 3H labelled cyclic AMP was obtained from New England Nuclear. RESULTS
The clinical definition of the cases and the histologic study of carcinomas and nodules are respectively presented in the left part of Table 1 and in Table 2. Among the 7 thyroid carcinomas, the histological aspect is considered as homogeneous in 4 cases and heterogeneous in 3 cases. Among the latter, it is not surprising to see the two medullary carcinomas which are known to present varying structure. The cell population is high to variable in 6 cases and is low in only one. Such findings are consistent with the reduced colloid space partially replaced by cell proliferation and stroma [12], as in experimental models [13]. Except in one case, a good correlation was obtained in double blind between the estimation of the cells: colloid ratio by histologic examination and biochemical DNA measurement. In the 6 hyperfunctional nodules, 3 were classified as heterogeneous and 3 as homogeneous. The cell density was more variable than in the carcinomas and ranged from average to low values. This fact is explained by the similarity between nodular and normal thyroid tissue, but also by marked structural changes induced by colloid deposits and cysts frequently encountered in goitrous disease. Here also a good correlation has been obtained between
450
G. Sand, A. Jortay, R. Pochet and J . E. Dumont
histologic examination and biochemical DNA measurement. The absolute values of DNA and protein content, and basal and stimulated adenylate cyclase and protein phosphokinase activities in carcinomas, hyperfunctional nodules and normal thyroid glands are presented in Tables 1, 3 and 4. As in other studies of this type [14], biochemical levels are highly variable from one case to another. However, it can be observed that the protein: DNA ratio is similar in all types of glands. With regard to the absolute levels of protein phosphokinase activi15
>-
O 09 IIII--
O>_
mE <~cn
--J l:g I.O 123
NORMAl_
NODULES
CARCINOMAS
Fig. 1. Adenylate cydase activity in normal thyroid tissue, autonomous nodules and carcinomas. Mean value + SEM. Arbitrary units are pmoles of cyclic A M P formed/10 min./#g DNA at 30°C with basal activity taken equal to I.
ties, activaty is significantly higher in several carcinomas, by a factor of up to 4-fold. Indeed, basal protein kinase specific activity (expressed in picomoles of phosphate incorporated into protein/rain/rag DNA) in carcinomas is similar to cyclic AMP-stimulated protein kinase activity in normal glands. Activation by cyclic A M P was similar in carcinomas, nodules, and normal glands homogenates; however in one case of papillary carcinoma, the activation factor reached a relatively high value. Adenylate cyclase basal activity was rather similar in normal, nodular and cancerous tissue. The stimulation factor by T S H was also similar. Freezing did not seem to affect TSH stimulation (Table 5). The effect of adrenalin was irregularly obtained and not statistically significant in the three series. A significant effect
of prostaglandin E 1 was only observed on the nodular adenylate cyclase. In this tissue the stimulation by sodium fluoride was also markedly enhanced compared to normal tissue.
DISCUSSION Studies of fibroblasts and transformed cell lines in culture have led to the general conclusion that there is an inverse relation between intracellular cAMP levels and growth, and that the characteristics of transformed cells are secondary to a decrease in the cell concentration of this nucleotide [1, 2]. However two facts are not easily reconciliable with this generalization: (1) tropic hormones enhance the accumulation of cAMP in their target tissues and promote growth; (2) cAMP levels in rapidly growing animal tumors are often high. It was therefore of interest to measure the activity of enzymes of the cAMP system in h u m a n tumors. Previous studies on adenylate cyclase in medullary carcinomas [15] and in two functioning h u m a n thyroid adenomas [16] did not demonstrate a clear cut relation between adenylate cyclase activity and its stimulation by T S H and the degree of malignancy. However, medullary carcinomas cannot be considered strictly as thyroid tumors, because this cell type is not implicated in thyroid hormone synthesis. These results have been confirmed in this study. There is no clear cut difference in adenylate cyclase activity and its activation by effectors such as TSH, sodium fluoride or prostaglandin between normal and carcinomatous tissue. Thus, so far as it can be evaluated on the limited samples of h u m a n tumors, these data do not support the hypothesis of an inverse relation between adenylate cyclase activity and malignancy. In contrast, in the hyperfunctional tissue of the autonomous nodules, the activation of adenylate cyclase by prostaglandin E~ and sodium fluoride was increased by a factor of 2. In some nodules adenylate cyclase has been shown to be more sensitive to T S H which could explain their growth and hyperfunctioning [ 16]. The present data suggest that increased sensitivity to other effectors might also play a role in the pathogenesis of these lesions. The low or absent adenylate cyclase activation in some normal and cancerous tissues does not fit in well with the previously observed stimulation of several TSH-dependent metabolisms in h u m a n normal thyroid tissue [17].
451
Adenylate Cyclase and Protein Phosphokinase Activities in Human Thyroid Table 3.
Biochemical results obtained on hyperfunctional nodules. Units: see legend of Table 1 Adenylate cyclase
Protein phosphokinase
Patient
DNA
Prot.
Basal
+ TSH
+ NaF
+ Adr.
+ PGEa
Basal
+ cAMP
J.C. M.F. J.W. C.E. M.I. M.D.
1.9 0-9 1.5 1.4 1.3 3.1
94 86 57 74 33 86
1.3 3-1 0.5 2.4 2.7 .
2.3 5.4 1"2 5.0 3.5
20.4 49.0 4-0 44.0 20.0 .
1.3 3.9 0.6 4.2 2.4
4.8 10.0 0.8 8-6 3.6
138 619 63 686 1,505 645
304 826 109 752 1,500 710
Table 4.
.
.
.
Biochemical results obtained on normal glands. Units: see legend of Table 1 Adenylate cyclase
Patient
DNA
Prot.
Basal
+ TSH
+ NaF
J.M. W.S. J.C. J.W. M.S. A.M. M.I. R.V. M.D.
1.0 0.8 1.8 1.8 1.4 2.7 1.6 0.9 2.4
291 88 119 79 57 73 55 38 139
1.0 2-1 1.0 2.8 2.1 1.1 5-9 3.3 .
2.7 3.2 1.3 3.5 5.6 1.4 5.9 2.9
6.4 . 6-0 7.8 13.4 23.0 41.0 17.1 .
Table 5.
.
.
Protein phosphokinase
+ Adr. 1
.
. 1.2 4.4 1.9 0.7 5.1 2.8
+ PGEx
Basal
+ cAMP
1.5 . 1.2 3.5 3.1 2-0 7.2 5.8
480
880 974 216 292 696 454 942 474 750
142 246 454 386 596 400 292
.
Comparison of adenylate cydase and protein phosphokinase activities in fresh and frozen tissue. Units: see legend of Table 1
Adenylate cyclase Activity
Fresh tissue sample
Frozen tissue sample
1
2
3
Mean Value
1
2
3
Mean Value
Bas~
1.6
1.6
1.5
1.6
1.7
2.1
1.6
1.8
+TSH TSH/Basal
2.5 1.6
3.2 2"0
2.0 1.3
2.6 1.6
3.3 1-9
2.8 1.3
2.1 1-3
2.7 1.5
+NaF NaF/Basal
5.7 3.6
7.9 4.9
3.4 2-3
5.7 3.6
7.3 4.3
6.9 3.3
7.1 4.4
7-1 3.9
Protein kinase activity Basal + cyclic AMP
170 284
H o w e v e r sensitivity o f a d e n y l a t e cyclase to effectors is notoriously fragile a n d in this case c o u l d h a v e been lost because o f the freezing o f the tissue. H o w e v e r , a c o m p a r i s o n o f adenylate cyclase a n d p r o t e i n p h o s p h o k i n a s e activity in fresh a n d frozen n o r m a l t h y r o i d do n o t show significant differences. I f a d e n y l a t e cyclase activity seems not to be
178 281
related to the degree o f m a l i g n a n c y , the situation is n o t the same for protein p h o s p h o kinase activity. I n d e e d , a l t h o u g h absolute e n z y m a t i c levels were variable from case to case, basal a n d c A M P - s t i m u l a t e d activity were h i g h e r in c a r c i n o m a s t h a n in n o r m a l tissue b y a factor o f u p to 4-fold. Nevertheless, the activation by c A M P was the same in n o r m a l
452
G. Sand, A. Jortay, R. Pochet and J. E. Dumont
and pathologic glands. Granner [18] has also observed 3-fold higher basal protein phosphokinase activity in a cell line established from a rat hepatoma (HTC) than in normal liver, but in the crude extracts, c A M P was unable to activate the enzyme. Thus protein phosphokinase and probably cyclic A M P - d e p e n d e n t protein phosphokinase were found in greater amounts in several types of h u m a n thyroid carcinomas. However, one cannot exclude at the present stage of work the possibility of modifications of the enzyme(s) which would increase the affinity for protein substrates or the inactivation of the thermostable protein phosphokinase inhibitor [19], if present in the thyroid. In conclusion, whereas from extrapolation of in vitro work on transformed cells one would have expected to find decreased levels of
adenylate cyclase and decreased sensitivity to effectors in human thyroid carcinomas, no such change has been observed. However, the activity of protein phosphokinase, the enzyme of which cyclic A M P is an effector, has been found to be markedly increased. It would be therefore of interest to investigate the end result of the modulation of adenylate cyclase and protein phosphokinase activities, i.e. protein phosphorylation in normal and cancerous human tissue.
Acknowledgements--This work was supported by a grant of the Fonds Canc~rologique de la Caisse G6n6rale d'Epargne et de Retraite (Belgium). The authors wish to thank Dr. P. Dor for his collaboration in providing the thyroid tissue specimens, Dr. E. Schell-Frederick for help in revising the text and Ms. D. Legrand for the careful typing of the manuscript.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 1 l.
12.
13.
W . L . RYAN and M. L. HEIDRICK, Role of cyclic nucleotides in Cancer. In Advances in Cyclic Nucleotide Research. (Edited by P. GR~ENGARD and G. A. ROBISON), Vol. 4, p. 81, Raven Press, New York (1974). J. OTT~N, G. S. JOHNSON and I. PASTAN, Cyclic AMP levels in fibroblasts: relationship to growth rate and contact inhibition of growth. Biochem. biophys. Res. Comm. 44, 1192 (1971). J . E . DUMONT, The action of thyrotropin on thyroid metabolism. In Vitamins and Hormones. Vol. 29, p. 287 Academic Press, New York (1971). S . C . WERNER and M. SPOONER, New and simple test for hyperthyroidism employing L-triiodothyronine and the twenty-four hour 13aI uptake method. Bull. N.Y. Acad. Press 31, 137 (1955). J . M . MILLERand J. I. HAMBURGER,The thyroid scintigram. I. The hot nodule. Radiobiology 84, 66 (1965). O . H . LOWRY,N. ROSEBROUGH,A. L. FARR and R. J. RANDALL,Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265 (1951). K. BURTON, Determination of DNA concentration with diphenylamine. In Methods in Enzymology. (Edited by L. GROSSMANand K. MOLDAVE), Vol. 12b, p. 163 Academic Press, New York (1968). g . POCHET, J. M. BOEYNAEMSand J. E. DUMONT, Stimulation by thyrotropin
of horse thyroid plasma membranes adenylate cyclase: evidence of cooperativity. Biochem. biophys. Res. Comm. 58, 446 (1974). J. RAMACHANDRAN,A new simple method for separation of adenosine 3'-5'cyclic monophosphate from other nucleotides and its use in the assay of adenyl cyclase. Analyt. Bioehem. 43, 227 (1971). I . M . GLYNN and J. B. CHAPPELL,A simple method for the preparation of 32p-labelled adenosine triphosphate of high specific activity. Biochem. J. 90, 147 (1964). D . A . WALSH, J. P. PERKINS, CH. O. BROSTROM, E. S. H o and E. G. KREBS, Catalysis of the phosphorylase kinase activation reaction. J. biol. Chem. 246, 1968 (1971). W . O . RUSSELL, M. L. IBANEZ, R. L. CLARK, C. S. HILL JR and E. C. WHITE, Follicular (organoid) carcinoma of the thyroid gland. Report of 84 cases. In Thyroid Cancer. UICC Monograph. (Edited by CH.E. HEDINGER).gol. 12, p. 14 Springer-Verlag, Berlin (1969). J. MATOVINOVIC, R. D. HILBERT, W. F. ARMSTRONGand R. G. HELOENSEN, Thyroid tumor and thyroid transplant tumor in iodine-deficient rat, In Current Topics in Thyroid Research. (Edited by CASSANO-ANDREOLI). p. 920 Academic Press, New York (1965).
Adenylate Cyclase and Protein Phosphokinase Activities in Human Thyroid 14. 15.
16. 17. 18. 19.
J . E . DUMONT,Carbohydrate metabolism in the thyroid gland. J. din. Endocr. 20, 1246 (1960). F. DE RUBERTIS,g . YAMASmTA, A. DEKKER, P. R. LARSENand J. B. FIELD, Effects of thyroid-stimulating hormone on adenyl cyclase activity and intermediary metabolism of "cold" thyroid nodules and normal human thyroid tissue, or. clin. Invest. 51, 1109 (1972). P . R . LARSEN,K. YAMASHITA,A. DEKKERand J. B. FIELD, Biochemical observations in functioning human thyroid adenomas, d. clin. Endocr. 36, 1009 (1973). J. OTTEN and J. E. DUMONT, Glucose metabolism in normal human thyroid tissue in vitro. Europ. d. din. Invest. 2, 213 (1972). D . K . GRANNER, Protein kinase: altered regulation in a hepatoma cell line deficient in adenosine 3',5'-cyclic monophosphate-binding protein. Biovhem. biophys. Res. Comm. 46, 1516 (1972). D.A. WALSH, CH. D. ASHBY,C. GONZALEZ,D. CALKINS,E. H. FISCHERand E. G. KR~Bs, Purification and characterization of a protein inhibitor of adenosine 3'-5'-monophosphate-dependent protein kinases. J. biol. Chem. 246, 1977 (1971).
453
Adenylate Cyclase and Protein Phosphokinase Activities in Human Thyroid. Comparison of Normal Glands, Hyperfunctional Nodules and Carcinomas* G. SAND~', A. JORTAY++, R. POCHET~ and J. E. DUMONT~ ~Institut de Recherche Interdisciplinaire, Facultg de Mgdecine, Universitd de BruxeUes and Biology Department§, Euratom, and +Service de Chirurgie de l'Institut Bordet, B-I O00 BruxelIes, Belgium A b s t r a c t - - B a s a l and stimulated adenylate cyclase and protein phosphokinase activities
were measured in normal and diseased human thyroids. Seven specimens of thyroid carcinomas (2 papillary, 2 mixed papillo-follicular or trabecular, 1 follicular and 2 medullary), and 6 hyperfunctional autonomous nodules were obtained at thyroidectomy. Nine specimens of normal tissue, taken from the opposite lobe of the nodular thyroids and from entirely normal glands, constituted the control group. Biochemical observations were the following: adenylate cyclase: basal activity and stimulation by T S H were rather similar in all cases. Hyperfunctional nodules were characterized by a significant increase of stimulation, as compared to the normal, by sodium fluoride (13'0 vs 7"S fold) and prostaglandin E x (2'6 vs l'3 fold). Adrenalin had no significant effect. Protein phosphokinase: basal activity was significantly higher in carcinomas by a factor of up to 4-fold. Activation by cyclic A M P was similar in normal and pathological homogenates. These data show that adenylate cyclase activity is not unequivocally related to the degree of malignancy in human thyroid tumors and that protein phosphokinase activity is increased in such tissues. The data are discussed within the framework of current hypotheses derived from studies of transformed cells in vitro.
INTRODUCTION
resulting in a decrease of cyclic A M P concentrations in transformed cells [1]. However, in apparent contradiction with this observation, cyclic A M P levels in rapidly growing animal tumors are high [1] and tropic hormones, such as thyrotropin (TSH), raise cyclic A M P levels and activate growth in their target tissues [3]. The aim of the present work was to compare the activity of two key enzymes of the cyclic A M P system: adenylate cyclase and cyclic AMP-dependent protein phosphokinase in normal and cancerous h u m a n thyroid tissue, and in autonomous hyperfunctioning nodules.
TH~ CYCLmA M P (adenosine 3',5'-cyclic monophosphate) system has been shown to be one of the major intracellular systems for relaying a hormone message from the plasma membrane to the nucleus. It has been implicated in cell differentiation and growth in in vitro preparations [1]. In fibroblasts in culture there is an inverse relation between growth and intracellular levels of cyclic A M P [2]. Moreover an essential event in transformation appears to be a depression in adenylate cyclase activity Accepted 15 January 1976. *This work was supported by a grant from the "Fonds Cancdrologique de la Caisse Gdndrale d'Epargne et de Retraite", Belgium. §Publication No. BIO 1200.
MATERIAL A N D M E T H O D S
Seven specimens of thyroid carcinomas were obtained from patients at thyroidectomy. There 447
448
G. Sand, A. Jorlay, R. Pochet and dr. E. Dumont
were 2 p a p i l l a r y , 2 m i x e d papillo-follicular or trabecular, 1 follicular a n d 2 m e d u l l a r y c a r c i n o m a s . T h e patients r a n g e d in age f r o m 7 to 66; 5 w e r e f e m a l e a n d 2 w e r e m a l e . N o n e o f these p a t i e n t s h a d received previous neck i r r a d i a t i o n . 131I iodide u p t a k e s w e r e w i t h i n n o r m a l limits in all p a t i e n t s a n d t h y r o i d scans u n i f o r m l y d e m o n s t r a t e d low or absence o f activity o v e r t u m o r sites. Six h y p e r f u n c t i o n a l nodules c h a r a c t e r i z e d b y elevated 131I iodide u p t a k e ( 5 0 % at the 6 t h h r ) a n d m a r k e d h y p e r a c t i v i t y at scan were excised. I n 3 cases, the T a suppression test a n d T S H s t i m u l a t i o n o f the s u r r o u n d i n g tissue c o n f i r m e d the diagnosis (see T a b l e 2) [4, 5]. All p a t i e n t s were f e m a l e a n d r a n g e d in age f r o m 33 to 40. N i n e specimens o f n o r m a l tissue t a k e n f r o m the opposite lobe o f the n o d u l a r thyroids a n d f r o m entirely n o r m a l glands constituted the control group. F o r n o r m a l thyroids a n d h y p e r f u n c t i o n a l
nodules, the v a s c u l a r s u p p l y was i n t e r r u p t e d 3 m i n or less before the s p e c i m e n was excised. T h i s t i m e i n t e r v a l was c o n s i d e r a b l y longer for c a r c i n o m a s a n d r a n g e d f r o m 10 to 30 min. T h e excised tissue was i m m e d i a t e l y p l a c e d in n o r m a l saline solution at 0°C, t r a n s p o r t e d to the l a b o r a t o r y , a n d frozen in liquid n i t r o g e n until the t i m e o f b i o c h e m i c a l m e a s u r e m e n t s . I n 1 e x p e r i m e n t with n o r m a l thyroid, the tissue was cut in 6 samples, 3 were assayed freshly a n d the 3 others w e r e frozen. Before freezing, a p o r t i o n was t a k e n for histologic e x a m i n a t i o n in s t a n d a r d h e m a t o x y l i n a n d eosin sections; 15 different fields d i s t r i b u t e d in the 3 m o s t r e p r e s e n t a t i v e sections o f each s p e c i m e n were observed. T h e s t r u c t u r a l p a t t e r n o f t h y r o i d tissue was defined as either h o m o g e n e o u s or heterogeneous after c o m p a r i s o n o f nodules a n d c a r c i n o m a s with n o r m a l thyroids. Criteria used were the r e g u l a r i t y o f follicle size, presence o f colloid cysts, a m o u n t o f c o n n e c t i v e tissue a n d
Table 1. Histologic and biochemical results obtained on thyroid carcinomas. DNA and protein content are expressed in mg/g wet tissue, adenylate cyclase activity in picomoles cyclic A M P formed/lO min/#g DNA, and protein phosphokinase activity in picomoles of phosphate incorporated into protein/min/mg DNA. Effectors added: TSH: 10 mU/ml ; sodium fluoride ( NaF) : 1"10 - 2 M ; adrenalin: 5"10 - 4 M ; prostaglandin E a : 5pg/ml ; cyclic A M P : 2"10 . 6 M Adenylate cyclase Carcinoma
Structural aspect
Cells Colloid
M.V. M.S. W.S. A.R. G.S.
Papillary Papillary Mixed Mixed Follicular
Homog. Homog. Homog. Homog. Heterog.
High Variable High High High
2.8 1.6 0.9 3.5 2-7
134 71 68 152 172
8.9 2.4 1.9 1.4 --
J.M. S.V.
Medullary Heterog. Medullary Heterog.
Low Variable
0.6 1.6
40 64
1.0 .
Patient
Pro+ DNA teins Basal TSH
.
+ NaF
11.0 4.5 2.1 2"8 1.6
60.0 16.7 -12.5 22.5
1"2
5.8 .
.
Protein phospho+ + kinase Adren. PGE I Basal +cAMP 14.6 2.1 -1.7 2.5
-4.0 -3.3 5.0
916 446 934 481 2,120
1.1
1.2
1,015 1,355 1 , 2 5 0 1,510
.
1,125 1,325 1,540 494 3,750
Table 2. Clinical definition and histological results of hyperfunctional nodules. PB 1271 is expressed in pg °/oo, 1 3xi uptake in ~ of tracer dose and PB 131I in mg/L plasma. T3 suppression: 0 = no suppression; - - not done
131I Uptake 6th hr 24th hr
TSH activation of T3 Sup- surrounding Pathological PB131I pression tissue findings
Patient
pB127I
J.C. M.F. J.W.
5.5 I 1.7 5" 1
28.9 63.8 53.9
42.5 60-3 78.8
0.30 0-35 0.49
--0
--+
C.E.
9-5
--
70.0
0"48
--
--
M.I. M.D.
6.5 7.7
--69.6
69.0 76.5
0" 14 1.54
-0
+ +
Structural aspect
\ Cylindrical Heterog. J epithelium Heterog. ] Colloid Homog. Goiter Homog. (Flattened epithelium) Homog. Colloid Heterog. cysts
Population cells~colloid Variable Low Average Low Low Variable
Adenylate Cyclase and Protein Phosphokinase Activities in Human Thyroid fibrosis or degenerative changes disrupting the basic follicular organization. Cell density was roughly evaluated using similar criteria applied to normal and pathological tissues. The cell population was expressed as low or average (typical of normal thyroid) or as high when colloid material was almost totally replaced by cell proliferation (mainly in carcinomas), or as variable if cell population varied greatly from one area to another. From 3 days to 2 months after excision, the frozen glands were thawed, rapidly trimmed free of fat and surrounding connective tissue, sliced with a Stadie Riggs microtome, chopped with a MacIlwain slicer, and homogenized with a motor driven teflon glass homogenizer by 4 strokes at a low speed in ice-cold medium (0"25M sucrose, 0"05M tris-HC1 buffer, pH 7"4). The preparation was filtered through a teflon sieve and used for enzymatic assays (homogenate). Protein content was assayed by the method of Lowry et al. [6] with bovine serum albumin as standard, and DNA by the method of Burton [7]. Adenylate cyclase was measured as previously described [8]. Briefly, aliquots of the homogenate were incubated 10 rain at 30°C in a medium (200/~1) containing 50 m M tris-HC1 buffer, pH 7"4, 2 m M cyclic AMP, 5 m M MgC12, 3 m M ATP, 5/~Ci a32p-ATP (Radiochemical Centre, Amersham, U.K.), 0"1% cristalline bovine serum albumin, 1 0 m M creatine phosphate and 60 pg cristalline rabbit muscle creatine kinase. At the end of the incubation, the test tubes were transferred to an ice bath and 100 pl of an ice-cold solution containing 20 m M cyclic AMP, 50 m M ATP and 3H-cyclic AMP (20,000 counts/rain) were added. Cyclic AMP was then isolated by elution on dry neutral alumina columns [9]. The eluate was diluted in Bray's solution and 3H and a2p radioactivities were counted in a liquid scintillation counter. Protein phosphokinase activity was assayed in a total volume of 1 ml: the complete reaction mixture contained enzyme plus 0"1 M potassium phosphate buffer pH 6"5, 1 m M EDTA, 1 0 m M magnesium acetate, 1 0 m M sodium fluoride, 2 m M theophylline, 2 mg of a mixture of histones (from calf thymus type II, Sigma), 0"2 m M ~-32p-ATP (15-20 dis. min-1/p mole) and, when added, 2 pM cyclic AMP. Assays were carried out at 30°C. After 4 min preincubation, the reaction was initiated by the addition of ~-32p-ATP and stopped after 30 sec, 1 min, 2 min and 4 min by the addition
449
of 1 ml of 40% trichloroacetic acid (TCA). The TCA precipitable material was washed two times by dispersion in cold N a O H 1N and reprecipitation in T C A 200/o. The final TCA precipitate was digested in 1 ml soluene-100 (Packard, Warenville, Ill., U.S.A.) and counted in toluene-omnifluor 4% (NEN, Boston, Mass., U.S.A.) with a Nuclear Chicago Mark II liquid scintillation counter (efficiency of 8090% for 32p). Enzyme activity was linear with time up to 4 m i n and up to a protein concentration of 1 mg. Cyclic AMP, ATP, phosphocreatine, creatine kinase and histones were purchased from Sigma (Saint Louis, Mo., U.S.A.), and adrenalin from Parke-Davis. Other reagents were of analytical grade (Merck, Darmstadt, F.R.D.). Bovine thyrotropin was obtained from Armour and from the N.I.H. Prostaglandin E t w a s a generous gift of Dr. J. Pike, Upjohn Co., Kalamazoo, Michigan, U.S.A. y-32p-ATP was prepared according to the method of Glynn and Chappell [10] modified by Walsh et al. [11], or was obtained from the Radiochemical Centre (Amersham, Great Britain). 3H labelled cyclic AMP was obtained from New England Nuclear. RESULTS
The clinical definition of the cases and the histologic study of carcinomas and nodules are respectively presented in the left part of Table 1 and in Table 2. Among the 7 thyroid carcinomas, the histological aspect is considered as homogeneous in 4 cases and heterogeneous in 3 cases. Among the latter, it is not surprising to see the two medullary carcinomas which are known to present varying structure. The cell population is high to variable in 6 cases and is low in only one. Such findings are consistent with the reduced colloid space partially replaced by cell proliferation and stroma [12], as in experimental models [13]. Except in one case, a good correlation was obtained in double blind between the estimation of the cells: colloid ratio by histologic examination and biochemical DNA measurement. In the 6 hyperfunctional nodules, 3 were classified as heterogeneous and 3 as homogeneous. The cell density was more variable than in the carcinomas and ranged from average to low values. This fact is explained by the similarity between nodular and normal thyroid tissue, but also by marked structural changes induced by colloid deposits and cysts frequently encountered in goitrous disease. Here also a good correlation has been obtained between
450
G. Sand, A. Jortay, R. Pochet and J . E. Dumont
histologic examination and biochemical DNA measurement. The absolute values of DNA and protein content, and basal and stimulated adenylate cyclase and protein phosphokinase activities in carcinomas, hyperfunctional nodules and normal thyroid glands are presented in Tables 1, 3 and 4. As in other studies of this type [14], biochemical levels are highly variable from one case to another. However, it can be observed that the protein: DNA ratio is similar in all types of glands. With regard to the absolute levels of protein phosphokinase activi15
>-
O 09 IIII--
O>_
mE <~cn
--J l:g I.O 123
NORMAl_
NODULES
CARCINOMAS
Fig. 1. Adenylate cydase activity in normal thyroid tissue, autonomous nodules and carcinomas. Mean value + SEM. Arbitrary units are pmoles of cyclic A M P formed/10 min./#g DNA at 30°C with basal activity taken equal to I.
ties, activaty is significantly higher in several carcinomas, by a factor of up to 4-fold. Indeed, basal protein kinase specific activity (expressed in picomoles of phosphate incorporated into protein/rain/rag DNA) in carcinomas is similar to cyclic AMP-stimulated protein kinase activity in normal glands. Activation by cyclic A M P was similar in carcinomas, nodules, and normal glands homogenates; however in one case of papillary carcinoma, the activation factor reached a relatively high value. Adenylate cyclase basal activity was rather similar in normal, nodular and cancerous tissue. The stimulation factor by T S H was also similar. Freezing did not seem to affect TSH stimulation (Table 5). The effect of adrenalin was irregularly obtained and not statistically significant in the three series. A significant effect
of prostaglandin E 1 was only observed on the nodular adenylate cyclase. In this tissue the stimulation by sodium fluoride was also markedly enhanced compared to normal tissue.
DISCUSSION Studies of fibroblasts and transformed cell lines in culture have led to the general conclusion that there is an inverse relation between intracellular cAMP levels and growth, and that the characteristics of transformed cells are secondary to a decrease in the cell concentration of this nucleotide [1, 2]. However two facts are not easily reconciliable with this generalization: (1) tropic hormones enhance the accumulation of cAMP in their target tissues and promote growth; (2) cAMP levels in rapidly growing animal tumors are often high. It was therefore of interest to measure the activity of enzymes of the cAMP system in h u m a n tumors. Previous studies on adenylate cyclase in medullary carcinomas [15] and in two functioning h u m a n thyroid adenomas [16] did not demonstrate a clear cut relation between adenylate cyclase activity and its stimulation by T S H and the degree of malignancy. However, medullary carcinomas cannot be considered strictly as thyroid tumors, because this cell type is not implicated in thyroid hormone synthesis. These results have been confirmed in this study. There is no clear cut difference in adenylate cyclase activity and its activation by effectors such as TSH, sodium fluoride or prostaglandin between normal and carcinomatous tissue. Thus, so far as it can be evaluated on the limited samples of h u m a n tumors, these data do not support the hypothesis of an inverse relation between adenylate cyclase activity and malignancy. In contrast, in the hyperfunctional tissue of the autonomous nodules, the activation of adenylate cyclase by prostaglandin E~ and sodium fluoride was increased by a factor of 2. In some nodules adenylate cyclase has been shown to be more sensitive to T S H which could explain their growth and hyperfunctioning [ 16]. The present data suggest that increased sensitivity to other effectors might also play a role in the pathogenesis of these lesions. The low or absent adenylate cyclase activation in some normal and cancerous tissues does not fit in well with the previously observed stimulation of several TSH-dependent metabolisms in h u m a n normal thyroid tissue [17].
451
Adenylate Cyclase and Protein Phosphokinase Activities in Human Thyroid Table 3.
Biochemical results obtained on hyperfunctional nodules. Units: see legend of Table 1 Adenylate cyclase
Protein phosphokinase
Patient
DNA
Prot.
Basal
+ TSH
+ NaF
+ Adr.
+ PGEa
Basal
+ cAMP
J.C. M.F. J.W. C.E. M.I. M.D.
1.9 0-9 1.5 1.4 1.3 3.1
94 86 57 74 33 86
1.3 3-1 0.5 2.4 2.7 .
2.3 5.4 1"2 5.0 3.5
20.4 49.0 4-0 44.0 20.0 .
1.3 3.9 0.6 4.2 2.4
4.8 10.0 0.8 8-6 3.6
138 619 63 686 1,505 645
304 826 109 752 1,500 710
Table 4.
.
.
.
Biochemical results obtained on normal glands. Units: see legend of Table 1 Adenylate cyclase
Patient
DNA
Prot.
Basal
+ TSH
+ NaF
J.M. W.S. J.C. J.W. M.S. A.M. M.I. R.V. M.D.
1.0 0.8 1.8 1.8 1.4 2.7 1.6 0.9 2.4
291 88 119 79 57 73 55 38 139
1.0 2-1 1.0 2.8 2.1 1.1 5-9 3.3 .
2.7 3.2 1.3 3.5 5.6 1.4 5.9 2.9
6.4 . 6-0 7.8 13.4 23.0 41.0 17.1 .
Table 5.
.
.
Protein phosphokinase
+ Adr. 1
.
. 1.2 4.4 1.9 0.7 5.1 2.8
+ PGEx
Basal
+ cAMP
1.5 . 1.2 3.5 3.1 2-0 7.2 5.8
480
880 974 216 292 696 454 942 474 750
142 246 454 386 596 400 292
.
Comparison of adenylate cydase and protein phosphokinase activities in fresh and frozen tissue. Units: see legend of Table 1
Adenylate cyclase Activity
Fresh tissue sample
Frozen tissue sample
1
2
3
Mean Value
1
2
3
Mean Value
Bas~
1.6
1.6
1.5
1.6
1.7
2.1
1.6
1.8
+TSH TSH/Basal
2.5 1.6
3.2 2"0
2.0 1.3
2.6 1.6
3.3 1-9
2.8 1.3
2.1 1-3
2.7 1.5
+NaF NaF/Basal
5.7 3.6
7.9 4.9
3.4 2-3
5.7 3.6
7.3 4.3
6.9 3.3
7.1 4.4
7-1 3.9
Protein kinase activity Basal + cyclic AMP
170 284
H o w e v e r sensitivity o f a d e n y l a t e cyclase to effectors is notoriously fragile a n d in this case c o u l d h a v e been lost because o f the freezing o f the tissue. H o w e v e r , a c o m p a r i s o n o f adenylate cyclase a n d p r o t e i n p h o s p h o k i n a s e activity in fresh a n d frozen n o r m a l t h y r o i d do n o t show significant differences. I f a d e n y l a t e cyclase activity seems not to be
178 281
related to the degree o f m a l i g n a n c y , the situation is n o t the same for protein p h o s p h o kinase activity. I n d e e d , a l t h o u g h absolute e n z y m a t i c levels were variable from case to case, basal a n d c A M P - s t i m u l a t e d activity were h i g h e r in c a r c i n o m a s t h a n in n o r m a l tissue b y a factor o f u p to 4-fold. Nevertheless, the activation by c A M P was the same in n o r m a l
452
G. Sand, A. Jortay, R. Pochet and J. E. Dumont
and pathologic glands. Granner [18] has also observed 3-fold higher basal protein phosphokinase activity in a cell line established from a rat hepatoma (HTC) than in normal liver, but in the crude extracts, c A M P was unable to activate the enzyme. Thus protein phosphokinase and probably cyclic A M P - d e p e n d e n t protein phosphokinase were found in greater amounts in several types of h u m a n thyroid carcinomas. However, one cannot exclude at the present stage of work the possibility of modifications of the enzyme(s) which would increase the affinity for protein substrates or the inactivation of the thermostable protein phosphokinase inhibitor [19], if present in the thyroid. In conclusion, whereas from extrapolation of in vitro work on transformed cells one would have expected to find decreased levels of
adenylate cyclase and decreased sensitivity to effectors in human thyroid carcinomas, no such change has been observed. However, the activity of protein phosphokinase, the enzyme of which cyclic A M P is an effector, has been found to be markedly increased. It would be therefore of interest to investigate the end result of the modulation of adenylate cyclase and protein phosphokinase activities, i.e. protein phosphorylation in normal and cancerous human tissue.
Acknowledgements--This work was supported by a grant of the Fonds Canc~rologique de la Caisse G6n6rale d'Epargne et de Retraite (Belgium). The authors wish to thank Dr. P. Dor for his collaboration in providing the thyroid tissue specimens, Dr. E. Schell-Frederick for help in revising the text and Ms. D. Legrand for the careful typing of the manuscript.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 1 l.
12.
13.
W . L . RYAN and M. L. HEIDRICK, Role of cyclic nucleotides in Cancer. In Advances in Cyclic Nucleotide Research. (Edited by P. GR~ENGARD and G. A. ROBISON), Vol. 4, p. 81, Raven Press, New York (1974). J. OTT~N, G. S. JOHNSON and I. PASTAN, Cyclic AMP levels in fibroblasts: relationship to growth rate and contact inhibition of growth. Biochem. biophys. Res. Comm. 44, 1192 (1971). J . E . DUMONT, The action of thyrotropin on thyroid metabolism. In Vitamins and Hormones. Vol. 29, p. 287 Academic Press, New York (1971). S . C . WERNER and M. SPOONER, New and simple test for hyperthyroidism employing L-triiodothyronine and the twenty-four hour 13aI uptake method. Bull. N.Y. Acad. Press 31, 137 (1955). J . M . MILLERand J. I. HAMBURGER,The thyroid scintigram. I. The hot nodule. Radiobiology 84, 66 (1965). O . H . LOWRY,N. ROSEBROUGH,A. L. FARR and R. J. RANDALL,Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265 (1951). K. BURTON, Determination of DNA concentration with diphenylamine. In Methods in Enzymology. (Edited by L. GROSSMANand K. MOLDAVE), Vol. 12b, p. 163 Academic Press, New York (1968). g . POCHET, J. M. BOEYNAEMSand J. E. DUMONT, Stimulation by thyrotropin
of horse thyroid plasma membranes adenylate cyclase: evidence of cooperativity. Biochem. biophys. Res. Comm. 58, 446 (1974). J. RAMACHANDRAN,A new simple method for separation of adenosine 3'-5'cyclic monophosphate from other nucleotides and its use in the assay of adenyl cyclase. Analyt. Bioehem. 43, 227 (1971). I . M . GLYNN and J. B. CHAPPELL,A simple method for the preparation of 32p-labelled adenosine triphosphate of high specific activity. Biochem. J. 90, 147 (1964). D . A . WALSH, J. P. PERKINS, CH. O. BROSTROM, E. S. H o and E. G. KREBS, Catalysis of the phosphorylase kinase activation reaction. J. biol. Chem. 246, 1968 (1971). W . O . RUSSELL, M. L. IBANEZ, R. L. CLARK, C. S. HILL JR and E. C. WHITE, Follicular (organoid) carcinoma of the thyroid gland. Report of 84 cases. In Thyroid Cancer. UICC Monograph. (Edited by CH.E. HEDINGER).gol. 12, p. 14 Springer-Verlag, Berlin (1969). J. MATOVINOVIC, R. D. HILBERT, W. F. ARMSTRONGand R. G. HELOENSEN, Thyroid tumor and thyroid transplant tumor in iodine-deficient rat, In Current Topics in Thyroid Research. (Edited by CASSANO-ANDREOLI). p. 920 Academic Press, New York (1965).
Adenylate Cyclase and Protein Phosphokinase Activities in Human Thyroid 14. 15.
16. 17. 18. 19.
J . E . DUMONT,Carbohydrate metabolism in the thyroid gland. J. din. Endocr. 20, 1246 (1960). F. DE RUBERTIS,g . YAMASmTA, A. DEKKER, P. R. LARSENand J. B. FIELD, Effects of thyroid-stimulating hormone on adenyl cyclase activity and intermediary metabolism of "cold" thyroid nodules and normal human thyroid tissue, or. clin. Invest. 51, 1109 (1972). P . R . LARSEN,K. YAMASHITA,A. DEKKERand J. B. FIELD, Biochemical observations in functioning human thyroid adenomas, d. clin. Endocr. 36, 1009 (1973). J. OTTEN and J. E. DUMONT, Glucose metabolism in normal human thyroid tissue in vitro. Europ. d. din. Invest. 2, 213 (1972). D . K . GRANNER, Protein kinase: altered regulation in a hepatoma cell line deficient in adenosine 3',5'-cyclic monophosphate-binding protein. Biovhem. biophys. Res. Comm. 46, 1516 (1972). D.A. WALSH, CH. D. ASHBY,C. GONZALEZ,D. CALKINS,E. H. FISCHERand E. G. KR~Bs, Purification and characterization of a protein inhibitor of adenosine 3'-5'-monophosphate-dependent protein kinases. J. biol. Chem. 246, 1977 (1971).
453