Modified cyclic nucleotide systems in Morris hepatoma 3924A favoring expression of cyclic GMP effect

419

Biochimica et Biophysica Acta, 500 (1977) 419--424 © Elsevier/North-Holland Biomedical Press

BBA 28345 MODIFIED CYCLIC NUCLEOTIDE SYSTEMS IN M O R R I S HEPATOMA 3924A F A V O R I N G EXPRESSION OF CYCLIC GMP E F F E C T

MAMORU SHOJI, HAROLD P. MORRIS a CRAIG W. DAVIS, NANCY L. BRACKETT and J.F. KUO Departments of Pharmacology and Medicine (Hematology and Oncology ), Emory University School of Medicine, Atlanta, Ga. 30322 and a Department o f Biochemistry, Howard University College o[ Medicine, Washington, D.C. 20059 (U.S.A.)

(Received April 4th, 1977)

Summary Modifications in the cyclic nucleotide systems favoring the expression of cyclic GMP effects were found to occur in the transplanted fast-growing Morris hepatoma 3924A. These included: ( a ) a decreased level of cyclic GMP phosphodiesterase and an increased level of cyclic AMP phosphodiesterase; (b) a disproportionately increased level of cyclic GMP-dependent protein kinase relative to that of cyclic AMP-dependent protein kinase; (c) a disproportionately increased level of stimulatory modulator of cyclic GMP
Introduction A number of recent experiments indicate that greatly elevated cellular contens of cyclic GMP, or increased ratios of cyclic GMP to cyclic AMP, are related to cell proliferation, exemplified by rapid growing hepatomas 3924A and 7288ctc [1], psoriatic lesions [2], adenocarcinoma of the human colon [3], renal cortical tumors [4], rapidly growing neonatal kidney [5] a n d adult kidney undergoing h y p e r t r o p h y [5]. A higher specific activity of guanylate cyclase has been reported in hepatoma 3924A [6], which in part could account for the elevated cyclic GMP level in the tumor. Even though increased cyclic AMP has been shown to be associated with decreased proliferation and increased "differentiation" in cell cultures [7], studies with the transplanted hepatoma show increased cyclic AMP compared to the liver [1,8]. It is interesting to note that urinary excretion of cyclic GMP increases in hepatomabearing rats, whereas urinary excretion cyclic AMP in the same animals is unaltered [9]. In the present study, we attempted to seek new information in

420 hepatoma 3924A concerning other parameters related to the accumulation as well as the actions of these two cyclic nucleotides, whose effects are shown to be mutually opposing in many instances [ 10]. Methods Hepatoma 3924A, 30 days after transplantation on ACI/c rats (female, about 140 g), were dissected free of necrotic tissues. The livers from the nontumor-bearing rats served as controls. The tissue samples (0.5--2 g) were quickly homogenized in 2 vol. ice-cold 50 mM Tris • HC1 buffer (pH 7.5) containing 3.75 mM mercaptoethanol (referred to as the extraction buffer), using glass-Teflon homogenizers. Crude extracts {soluble fraction) were obtained by centrifuging the homogenates for 20 min at 30 000 g. The standard assay system [11,12], for phosphodiesterases contained, in a volume of 0.1 ml, Tris • HC1 buffer (pH 8.0), 5.0 pmol; EDTA, 0.25 pmol; MgC12,2 pmol; snake venom (Crotalus adamanteus), 2 0 p g ; cyclic [G-3H]GMP or cyclic [G-3H]AMP, 0.1 nmol, containing a b o u t 1 • l 0 s cpm, and appropriate amounts of enzyme proteins (5--20 pg). The reaction was carried o u t at 37°C for 2--10 min so that the a m o u n t of substrate hydrolyzed was approx. 5--20% of that originally added to the incubation mixture. One unit of phosphodiesterase activity is defined as that a m o u n t of activity that hydrolyzes 1 pmol of cyclic nucleotide under the assay conditions. The standard assay system [13,14] for protein kinases contained, in a volume of 0.2 ml, potassium phosphate buffer (pH 7.0), 10 pmol; theophylline, 0.5 pmol; arginine-rich histone (HA, Worthington), 20 pg; MgC12, 2 pmol, [~/-~2P]ATP, 1 nmol, containing about 1 . 5 . 1 0 6 cpm; either cyclic GMP or cyclic AMP, 80 pmol; appropriate amounts {10--28 pg) of enzyme proteins; and stimulatory modulators [13,14] of cyclic GMP-dependent protein kinase, 35--40 pg. Since the stimulatory modulator has no effect on cyclic AMP-dependent protein kinase, the present system is suitable for assaying for both classes of protein kinase in tissue extracts or crude enzyme preparations in the same incubation solution. The concentration (0.4 pM) of either cyclic nucleotide used was such that they activate preferentially the respective classes of protein kinase to a near maximal extent without significantly cross-activating the other. The reaction was carried o u t for 10 min at 30°C. One unit of the enzyme is that a m o u n t of activity that transfers 1 pmol of 32p from [~,-32P]ATP to histone under the assay conditions. Crude protein kinase modulator was prepared from tissue extracts by steps of heating and trichloroacetic acid precipitation, as described elsewhere [13--15]. The activity of stimulatory modulator in the crude protein kinase modulator preparations thus obtained was assayed on the basis of its ability to specifically stimulate cyclic GMP-dependent protein kinase [13], whereas the activity of inhibitory m o d u l a t o r was assayed on the basis of its ability to specificaUy inhibit cyclic AMP-dependent protein kinase [ 13--15], under the assay conditions for protein kinases. One unit of the stimulatory modulator is defined as that a m o u n t of activity that stimulates 20 units of cyclic GMPdependent protein kinase 20% in the presence of 0.5 pM cyclic GMP. One unit of the inhibitory modulator is defined as that a m o u n t of activity that inhibits 20 units of cyclic AMP-dependent protein kinase 20% in the presence of

421

0.5 pM cyclic AMP. The assay system for phosphoprotein phosphatase contained, in a volume of 0.2 ml, Tris-HC1 (pH 7.5), 10 pmol; [32p]histone, 40pg, containing 200pmol 32p and about 2 . l 0 s cpm; and appropriate amounts of tissue extracts (20--30 pg). The reaction was carried out for 10 min at 30°C. One unit of phosphatase is defined as that amount of activity that hydrolyzes 1 pmol of 32p from [32P]histone under the incubation conditions. [32P]histone was prepared by phosphorylating mixed histone by excessive amounts of [7-32P]ATP and cardiac cyclic AMP
6[ LIVER_ 4L ~ CyclicAMP

g o

I ,~

-

--

^6MKC

' " ~

,

IO

i

20

30 40 50 60 FRACTION No.

70

80

Fig. 1. D E A E - c e l l u l o s e c h r o m a t o g r a p h y o f p h o s p h o d i e s t e r a s e s in e x t r a c t s o f t h e r a t liver a n d h e p a t o m a 3 9 2 4 A . E x t r a c t s (5 m l , e q u i v a l e n t t o 2 . 5 g f r e s h tissue) w e r e a p p l i e d t o D E A E - c e l l u l o s e c o l u m n s ( 1 . 5 X 17 c m ) . A f t e r t h e c o l u m n s w e r e w a s h e d ( f r a c t i o n s 1 - - 2 3 ) w i t h 1 3 0 m l o f t h e e x t r a c t i o n b u f f e r , t h e e n z y m e s w e r e e l u t e d ( f r a c t i o n s 2 4 - - 8 0 ) w i t h a KC1 l i n e a r g r a d i e n t ( 0 . 1 - - 0 . 6 M; t o t a l v o l u m e 1 2 0 m l ) , K C 1 b e i n g d i s s o l v e d in t h e s a m e b u f f e r , a c c o r d i n g t o t h e m e t h o d r e p o r t e d e l s e w h e r e [ 1 2 ] . A l i q u o t s (0.01 ml) of the fractions were assayed for the enzyme activities using 1 pM either cyclic GMP or cyclic A M P as s u b s t r a t e as d e s c r i b e d i n T a b l e I.

7.7 + 1.6 1 . 5 +, 0.1 *

Cyclic GMP

1 3 . 8 -+ 2.9 2 4 . 1 +_ 0 . 4 **

Cyclic AMP

Phosphodiesterase (units × 10-3/g tissue)

Significantly different from the liver: • P < 0.01; • * P < 0.05.

Liver Hepatoma 3924A

Tissue sample

activity/cyclic AMP-stimulated activity [16].

0.56 ± 0.02 0 . 0 6 +, 0 . 0 1 *

Activity ratio

A l l d a t a p r e s e n t e d are t h e m e a n s (+ s t a n d a r d e r r o r s ) o f t h e v a l u e s o b t a i n e d

KINASES STIMULATED

B Y C Y C L I C G M P A N D C Y C L I C AMP~

20.1 + 2.0 3 3 . 2 +- 2.1 **

Basal

24.1 + 2.0 4 1 . 1 +, 0.1 *

+ cyclic GMP

Protein kinase (units X 10-3/g tissue)

4 6 . 2 +_ 3 . 0 5 9 . 1 + 1 . 2 **

+ cyclic AMP 0 . 1 2 _+ 0 . 0 2 0 . 2 6 +- 0 . 0 2 **

Activity ratio

f r o m t h r e e r a t s . A c t i v i t y r a t i o o f p r o t e i n k i n a s e is d e f i n e d as c y c l i c G M P - s t i m u l a t e d

CONTENTS OF PHOSPHODIESTERASES FOR CYCLIC GMP AND CYCLIC AMP, PROTEIN IN E X T R A C T S O F T H E R A T L I V E R A N D H E P A T O M A 3 9 2 4 A

TABLE I

t~ b~

423

findings thus provide evidence, in addition to the increased particulate guanylate cyclase reported by others [6], to explain why the cyclic GMP is higher in the hepatoma than in the liver. We observed earlier that the foetal guinea pig liver and lung (tissues presumably undergoing rapid cell proliferation), have higher ratios of cyclic GMP to cyclic AMP hydrolysis than those seen in tissues from the adult animal [12]. This finding suggests some fundamental differences may exist between the pathological tumor growth and physiological tissue development; and changes in the phosphodiesterase pattern may constitute one of these differences. The protein kinase activity in extracts of hepatoma, assayed either in the absence or presence of added cyclic nucleotide (0.4 pM), were higher than that in the liver (Table I). The ratio of the cyclic GMP-stimulated activity to the cyclic AMP-stimulated activity, however, was slightly but significantly higher in hepatoma, suggesting that cyclic GMP-dependent protein kinase was relatively more abundant in the tumor. Although data are not shown, similarly altered patterns of phosphodiesterases and protein kinases shown for the soluble fraction from hepatoma (Table I) were also noted for the particulate fraction (30 000 × g pellets). Much higher ratios of cyclic GMP-dependent to cyclic AMP-dependent protein kinase were reported earlier for the foetal lung and heart of the guinea pig compared to those from the adult animal; developmental change in the ratio was, however, not observed for the liver [16]. Interestingly, the hypertrophied heart of the spontaneously hypertensive rat has a lower ratio of the protein kinases compared to that seen in the heart of the normotensive rat [17]. Most recently we noted that the ratio increases in the stressed and thickened vein of the dog created by side-by-side femoral arteriovenous fistulae [ 18]. Stimulatory modulator and inhibitory modulator, and their ratio, were higher in hepatoma than the liver (Table II). Similar findings were made earlier in the pancreas of the alloxan-induced diabetic rat [19], and in the enlarged lung (due to hypertrophy and hyperplasia) of the mouse following butylated hydroxytoluene administration (unpublished observation). The present findings clearly suggest that cyclic GMP-dependent protein kinase activity is enhanced in hepatoma, and that the cyclic AMP-dependent enzyme activity is suppressed,

TABLE lI CONTENTS EXTRACTS

OF PROTEIN KINASE MODULATORS AND OF THE RAT LIVER AND HEPATOMA 3924A

PHOSPHOPROTEIN

PHOSPHATASE

IN

A l l v a l u e s p r e s e n t e d are t h e m e a n s (-+ standard errors) o f t h e data o b t a i n e d f r o m s i x rats. Tissue sample

Protein kinase modulator ( u n i t s X 1 0 -3 g / t i s s u e )

A c t i v i t y ratio

Phosphoprotein phosphatase

Liver Hepatoma

3924A

Inhibitory

1.1 + 0 . 1 4 . 3 _+ 0 . 3 *

1 . 4 -+ 0 . 1 2 . 7 -+ 0 . 2 *

S i g n i f i c a n t l y d i f f e r e n t f r o m the l i v e r : * P < 0.01~ ** P < 0 . 0 5 .

0.83 + 0.09 1 . 6 0 +- 0 . 1 4 *

X 10-3/g

tissue)

(units/rag protein in e x t r a c t )

8 8 +- 1 3 88 + 15

7 2 9 _+ 1 0 9 1 4 1 8 -+ 2 4 0 **

(units

Stimulatory

424 when compared to normal liver. The net effect would be an imbalanced augmentation of cyclic GMP effects in hepatoma. This possibility appears to be greatly enhanced by the following observations made with hepatoma, namely: (a) high cyclic GMP content [1], a result of high guanylate cyclase [6] and low cyclic GMP-phosphodiesterase activity (Table I and Fig. 1), and (b) relative abundance of cyclic GMP-dependent protein kinase compared to the cyclic AMP-dependent enzyme (Table I). Hepatoma 3924A, therefore, may be characterized as a neoplastic tissue in which cyclic GMP effects are predominant. The level of phosphoprotein phosphatase, an enzyme that negates the phosphorylating actions of protein kinase, was higher in hepatoma than in the liver when the values were expressed on a basis of soluble protein in extracts (Table II). However, the difference disappeared when the values were expressed on a tissue weight basis. This is due to the fact that only 6% of hepatoma fresh weight is soluble protein compared to 12% of liver. In this regard, the differences observed for phosphodiesterase and protein kinase (Table I) and protein kinase modulators (Table II) for hepatoma would be greater if the values were expressed on a soluble protein basis. The significance of higher specific activity of the phosphatase in the hepatoma cytosol is not clear. It probably has a role in attenuating actions of cyclic AMP-dependent protein kinase, the dominant class of protein kinase, and thus, indirectly, helps to amplify cyclic GMPdependent protein kinase actions. Identification of cellular proteins phosphorylated by either class of protein kinase, presumably the ultimate determinates in sequences of events that initiate cyclic nucleotide formation in response to certain stimuli, is clearly in order. Acknowledgment This work was supported by grant HL-15969, and in part by grants CA-16255 and CA-10729 from USPHS. References 1 T h o m a s , E.W., M u r a d , F., L o o n e y , W.B. a n d M o r r i s , H . P . ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 2 9 7 , 5 6 4 - 567 2 V o o r h e e s , J., S t a w i s k i , M., Due[l, E., H a d d o x , M. a n d G o l d b e r g , N. ( 1 9 7 3 ) Life Sci. 1 3 , 6 3 9 - - 6 5 3 3 D e R u b e r t i s , F . R . , C h a y o t h , R . a n d F i e l d , J.B. ( 1 9 7 6 ) J, Clin. I n v e s t . 5 7 , 6 4 1 - - 6 4 9 4 Criss, W., M u r a d , F. a n d K i m t t r a , H . ( 1 9 7 6 ) J. C y c l i c N u c l e o t i d e Res. 2, 1 1 - - 1 9 5 S c h l o n d o r f t , D. a n d W e b e r , H. ( 1 9 7 6 ) P r o c . N a t l . A c a d , Sci. U.S. 7 3 , 5 2 4 - - 5 2 8 6 K i m u r a , H . a n d Murad~ F. ( 1 9 7 5 ) P r o c . N a t l . A c a d . Sci. U.S. 7 2 , 1 9 6 5 - - 1 9 6 9 7 R y a n , W,L. a n d H e i d r i c k , M . L . ( 1 9 6 8 ) S c i e n c e 1 6 2 , 1 4 8 4 - - 1 4 8 5 S C h a y o t h , R . , E p s t e i n , S. a n d F i e l d , J . B . ( 1 9 7 2 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 4 9 , 1 6 3 3 - - 1 6 3 9 9 M u r a d , F., K i m u r a , H., H o p k i n s , H., L o o n e y , W.B. a n d K o v a s s , C.J. ( 1 9 7 5 ) S c i e n c e 1 9 0 , 5 8 - - 6 0 1 0 G o l d b e r g , N . D . , H a d d o x , M . K . , N i c o l , S.E., Glass, D . B . , S a n f o r d , C . H . , K u e h l , J r . , F . A . a n d E s t e n s e n , R . ( 1 9 7 5 ) A d v . C y c l i c N u c l e o t i d e R e s . 5, 3 0 7 - - 3 3 0 11 T h o m p s o n , W.J. a n d A p p l e m a n , M.M. ( 1 9 7 1 ) B i o c h e m i s t r y 1 0 , 3 1 1 - - 3 1 6 1 2 Davis, C.W. a n d K u o , J . F . ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A e t a 4 4 4 , 5 5 4 - - 5 6 2 1 3 K u o , W.N. S h o j i , M. a n d K u o , J . F . ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 4 3 7 , 1 4 2 - - 1 4 9 1 4 K u o , W.N. a n d K u o , J . F . ( 1 9 7 6 ) J . Biol. C h e m . 2 5 1 , 4 2 8 3 - - - 4 2 8 6 1 5 W a l s h , D . A . , A s h b y , C.D., G o n z a l e z , C., C a l k i n s , D., F i s h e r , E . H . a n d K r e b s , E . G . ( 1 9 7 1 ) J . Biol. Chem. 246, 1977--1985 16 K u o , J . F . ( 1 9 7 5 ) P r o c . N a t l . A c a d . Sci. U.S. 7 2 , 2 2 5 6 - - 2 2 5 9 17 K u o , J . F . , Davis, C.W. a n d Tse, J. ( 1 9 7 6 ) N a t u r e 2 6 1 , 3 3 5 - - 3 3 6 1 8 K u o , J . F . , M a l v e a u x , E . J . , P a t r i c k , J . G . , Davis, C.W., K u o , W,-N. a n d P r u i t t , A.W. ( 1 9 7 7 ) B i o c h i m Biophys. Acta 497, 785--796 19 K u o , J . F . ( 1 9 7 5 ) B i o c h e m . B i o p h y s . Res, C o m m u n . 6 5 , 1 2 1 4 - - 1 2 2 0