35
Biochimica et Biophysica Acta, 497 ( 1 9 7 7 ) 3 5 - - 4 5 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 28196
ACTION OF CYCLIC NUCLEOTIDE ANALOGUES IN CHINESE HAMSTER OVARY CELLS
J. P A T R I C K O ' N E I L L , A L B E R T P. LI * a n d A B R A H A M W. HSIE
The University of Tennessee, Oak Ridge Graduate School of Biomedical Sciences and the Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 3 7830 (U.S.A.) (Received October 4th, 1976)
Summary Cyclic nucleotide analogues have been tested for their ability to cause the morphological conversion of Chinese hamster ovary cells in culture, as well as for effects on cyclic AMP-related enzymes. The ability of the analogues to inhibit the cyclic AMP phosphodiesterase activity and to activate the cyclic AMP-dependent protein kinase activity in cell extracts has been measured. Cell cultures were incubated with the analogues and the effects on morphology, intracellular level of cyclic AMP, and in vivo protein kinase activation were determined. All analogues which induced the morphological conversion also caused in vivo activation of the cyclic AMP-dependent protein kinase. Only N6,O2'-dibutyryl and N6-monobutyryl cyclic AMP caused on increase in intracellular cyclic AMP, presumably through inhibition of the intracellular cyclic AMP phosphodiesterase activity. The increase in cyclic AMP appears to cause the protein kinase activation. However, analogues such as 8-bromo and 8benzylthio cyclic AMP do not cause any change in intracellular cyclic AMP level and appear to activate the intracellular cyclic AMP-dependent protein kinase directly.
Introduction
Treatment of Chinese hamster ovary (CHO) cells with N6,O2'-dibutyryl adenosine 3' :5'-phosphate (dibutyryl cyclic AMP) causes the usually epitheliallike cells to convert to a fibroblast-like morphology [1--3]. This morphological conversion occurs in enucleated cells [4], shows cell cycle phase specificity [ 5], and involves the reorientation of microtubules [6]. * Present address: Cancer Research and T r e a t m e n t Center, University of N e w Mexico, Albuquerque, N.M., U.S.A.
36 The morphological conversion of CHO cells by dibutyryl cyclic AMP is a rather specific phenomenon. The conversion is not induced by adenine, adenosine, AMP, butyric acid, or unsubstituted cyclic AMP. The inactivity of cyclic AMP can be explained by its rapid degradation by the cyclic AMP phosphodiesterase activity in CHO cells [7]. O2'-Monobutyryl cyclic AMP is also inactive due to its rapid degradation by cyclic AMP phosphodiesterase as well as an O2'-deacylating activity in CHO cells [8]. N6-Mono butyryl cyclic AMP is active, probably due to its relative stability in CHO cells [7,8]. The treatment of actively growing CHO cells with dibutyryl cyclic AMP results in the intracellular accumulation of N6-monobutyryl cyclic AMP and an increase in the endogenous level of intracellular cAMP, presumably due to inhibition of the cyclic AMP phosphodiesterase activity by N6-monobutyryl cyclic AMP [7,8]. This treatment also results in the intracellular activation of the cyclic AMP-dependent protein kinase [9]. We have now extended these studies to other cyclic nucleotide analogues both for effects on properties of cells in culture (in vivo) and on the cyclic AMP-related enzymes in cell-free extracts (in vitro). The ability of these analogues to induce cell elongation, modify the intracellular level of cyclic AMP, and activate the cyclic AMPdependent protein kinase in vivo was investigated. Cell-free extracts were utilized to study the in vitro inhibition of cyclic AMP phosphodiesterase as well as activation of the cyclic AMP-dependent protein kinase. These studies were performed to further define the specificity of the morphological conversion of CHO cells and to attempt to discern the mechanism of action of cyclic nucleotide analogues in this system. The activation of the cyclic AMP-dependent protein kinase has been proposed to be the primary mechanism by which cyclic AMP effects are mediated [10,11]. The mechanism of action of cyclic nucleotide analogues is less clear, although protein kinase activation appears to be involved [9,12]. The analogues may act through inhibition of the cyclic AMP phosphodiesterase to cause an increase in endogenous cyclic AMP, which leads to protein kinase activation, or they may activate the protein kinase directly. In CHO cells the mechanism appears to be dependent on the cyclic nucleotide analogue utilized. Materials and Methods Cell culture. CHO cell clone CHO-K, was employed in these studies and has been described previously [1,13]. The cells were grown in medium F12 supplemented with 10% heat inactivated, dialyzed fetal calf serum (Pacific Biological Co., Richmond, Calif.) in plastic culture dishes under standard cell culture conditions of 5% CO2 in air at 37°C in a 100% humidified incubator. The morphological conversion of the CHO cells by cyclic nucleotides has been described previously [1--3]. Assay of cyclic AMP phosphodiesterase. The measurement of cyclic AMP phosphodiesterase activity in cell extracts has been described in detail previously [7]. The radiodisplacement assay of Brooker et al. [14] was employed, with slight modification, to measure the conversion of 3H-labeled cyclic AMP to adenosine. Several time points were taken with each concentration of sub-
37 strate and inhibitor, usually until 10% of the substrate had been hydrolyzed, to insure that the reaction rate is linear with time. The velocity of the reaction is expressed as pmol of adenosine formed per min per mg of protein. Assay o f cyclic AMP-dependent protein kinase and activation by cyclic nucleotide analogues in vitro. Protein kinase activities were measured by the incorporation of [32P]phosphate from [7-32P]ATP (New England Nuclear, Boston, Mass., > 1 2 Ci/mmol) into calf thymus histone mixture (Aldrich Chem. Co., Milwaukee, Wisc.). The assay developed by Corbin et al. [15] was followed except that 50 mM Tris • HC1 buffer (pH 7.5) was used instead of the 17 mM phosphate buffer (pH 6.8). Assays were performed in the presence or absence of different concentrations of cyclic AMP or other cyclic nucleotides as indicated. The velocity of the reaction is expressed as pmol of [32p]phosphate transferred to histone per min per mg protein. Measurement o f intracellular concentrations o f cyclic AMP. For the measurement of intracellular cyclic AMP, CHO cells were grown to a b o u t 50% confluency on 150-mm plastic culture dishes (Falcon, Oxnard, Calif.). Cultures were incubated with cyclic nucleotide analogues at a concentration of 1 mM in serum-containing media for 6 h. This is sufficient time for the morphological conversion to occur [1]. Since cyclic nucleotide analogues would be expected to interfere with the cyclic AMP-binding assay described below, several precautions were taken to ensure that a measurement of intracellular cyclic AMP was actually being obtained. As controls, plates were treated with medium containing the cyclic nucleotide analogue for approx. I s, that is, the minimum time taken to pour the medium on and off the plates. Both 6-h-treated and control plates were then washed two times with medium, followed by 10 washes with cold 0.15 M NaC1 in 0.01 M potassium phosphate (pH 7.0). These washes were necessary to remove the contaminating traces of media containing the cyclic nucleotide analogue. Untreated cultures were also washed in this manner and only a small decrease in measured steady-state level of cyclic AMP was found. This small effect is probably due to the use of cold saline for the washes which would minimize the energy-dependent efflux of cyclic AMP from the cells [16,17]. Trichloroacetic acid extracts were then made as described previously [7]. Although the medium contamination with cyclic nucleotide analogues was minimized, there was the additional problem of intracellular accumulation of these analogues. Therefore, the extracts were then chromatographed on D o w e x (Sigma Chemical Co., St. Louis, Mo., 50W, hydrogen form) columns to separate cyclic AMP from the analogues as described previously [7]. The elution profile of all the analogues was first determined so that the fractions collected contained only cyclic AMP with minimal contamination by the analogues. The a m o u n t of cyclic AMP was measured by the binding assay described previously [7]. Several dilutions of the extracts were assayed and the total intracellular cyclic AMP is expressed as pmol per 106 cells. Measurement o f the activation o f the cyclic AMP-dependent protein kinase in analogue-treated cells. Cultures were grown and washed exactly as described above for the measurement of intracellular cyclic AMP. The culture dishes were then frozen in liquid nitrogen, thawed in a buffer containing 50 mM Tris • HC1 (pH 7.5), 0.5 M NaC1, 10% glycerol, I mM EDTA, and 13.8 mM fl-mercaptoethanol, and homogenized with a Duall glass homogenizer. After centrifugation
38 at 20 000 X g for 15 min, the supernatant was assayed for protein kinase activity as described above. The assay was performed in the presence and absence of 1 pM cyclic AMP, a concentration which gives m a x i m u m activation of the enzyme and causes dissociation of the holoenzyme into subunits [9]. The ratio of activity in the absence of cyclic AMP to that found in the presence of cyclic AMP is expressed as the protein kinase activity ratio. The activity from untreated cultures is stimulated 3--4-fold by cyclic AMP and has an activation ratio of 0.25--0.35. Completely activated protein kinase would have an activation ratio of 1.0, as reported previously in dibutyryl cyclic AMP-treated cells [7]. Results
We have tested the ability of a number of cyclic nucleotide analogues at a concentration of 1 mM to induce the morphological conversion of CHO cells. We find that dibutyryl cyclic AMP, N6-monobutyryl cyclic AMP, 8-bromo cyclic AMP, and 8-benzylthio cyclic AMP do cause the cells to elongate. Unsubstituted cyclic AMP, O2'-monobutyryl cyclic AMP, cyclic GMP, N2,O2'-dibu tyryl cyclic GMP, and 8-bromo cyclic GMP are inactive in this system. To understand the mechanism by which the active analogues function and the reason for the inactivity of the others, we studied the ability of these analogues to mimic cyclic AMP in two enzymatic reactions (inhibition of cyclic AMP phosphodiesterase and activation of the cyclic AMP-dependent protein kinase). Previously, we reported two phosphodiesterase activities in CHO cells which differ in affinity for cyclic AMP [7,18]. One activity has a high apparent Km for cyclic AMP of 1--3 mM which appears to preclude a physiological role for this activity. Further study has revealed two activities which show low apparent
150-
K i n = 8 . 9 x 10 . 6
I00-
M
~\~
50.
.
o
,b
2'0
v/s
sb
4'0
F i g . 1. E a d i e - H o f s t e e g r a p h o f t h e k i n e t i c s o f d e g r a d a t i o n o f c y c l i c A M P b y c y c l i c A M P p h o s p h o d i e s t e r a s e i n C H O cell e x t r a c t s . A r a n g e o f c y c l i c A M P f r o m 6 X 1 0 - 8 t o 2 X 1 0 - 5 M w a s u s e d . The apparent K m v a l u e s f o r c y c l i c A M P a r e c a l c u l a t e d f r o m the slopes of the l i n e s .
39
0.5-
t/v
0.3.
0.1
10
20
10
20
0.5-
t/v
0.3
0.1
[INHIBITOR] x 10 - 5
Fig. 2. D i x o n g r a p h o f t h e k i n e t i c s o f i n h i b i t i o n o f c y c l i c A M P p h o s p h o d i e s t e r a s e b y N 6 - m o n o b u t y r y l cyclic A M P ( A ) a n d 8-benzylthio c y c l i c A M P (B). C y c l i c A M P w a s p r e s e n t a t 0.3 p M (~), 0 . 5 ~ M (D), 1 . 0 p M (A) a n d 3 . 0 p M (m). T h e a p p a r e n t K i values o f c o m p e t i t i v e i n h i b i t i o n are c a l c u l a t e d f r o m the p o i n t s o f i n t e r c e p t i o n o f t h e lines.
Km values for cyclic AMP (Fig. 1). The ability of the analogues to inhibit the hydrolysis of cyclic AMP in cell-free extracts was measured, and the data obtained with N 6 - m o n o b u t y r y l and 8-benzylthio cyclic AMP are shown in Fig. 2 as typical examples. The inhibition is competitive with respect to cyclic AMP, and two apparent Ki values of inhibition are obtained, presumably reflecting inhibition of both phosphodiesterase activities. The data obtained from these are summarized in Table I. All the cyclic AMP derivatives show two apparent K i values of inhibition. The two m o n o b u t y r y l derivatives and 8-benzylthio cyclic AMP are similar, with 8-bromo and dibutyryl cyclic AMP less effective inhibitors. Cyclic GMP and its t w o derivatives are the least effective inhibitors, and only one apparent K i of inhibition was found. This poor inhibition by cyclic GMP and its derivatives show the cyclic AMP specificity of the phosphodiesterase activities in CHO cells. These inhibition studies show that the cyclic AMP analogues apparently c o m p e t e with cyclic AMP for binding to the phosphodiesterase enzyme. However, the ability of the phosphodiesterase activities to hydrolyze the analogues
40 TABLE
I
INHIBITION LOGUES
OF CYCLIC
AMP
PHOSPHODIESTERASE
ACTIVITY
BY CYCLIC
NUCLEOTIDE
ANA-
The cyclic A M P phosphodiesterase activity in ceU-free extracts of C H O cells was assayed with radiolabeled cyclic A M P as substrate. For cyclic A M P the appaxent K m values are given; for the remainder of the cyclic nucleotides, the appaxent K i values of competitive inhibition are given. The values were calculated from the intercepts of the lines obtained on Dixon graphs. Cyclic nucleotide
Cyclic A M P
Cyclic A M P O2'-Monobutyryl cyclic A M P N 6 - M o n o b u t y r y l cyclic A M P N 6, O2'-Dibutyryl cyclic A M P 8-Benzyltbio cyclic A M P 8 - B r o m o cyclic AMP
1.2 1.1 3.5 2.3 4.0 8.3
cyclic GMP N 2, O 2 ' - D i b u t y r y l cyclic GMP 8 - B r o m o cyclic GMP
1.7 × 10 -3 1.9 X 10 -3 2.0 X 1 0 -3
X X X X × X
10 I0 10 10 10 10
-6 -s -5 -4 -5 -5
pbosphodiesterase activity (M)
8.9 2.5 1.5 7.1 2.1 4.2
× X X × X X
10 -6 10 -4 10 -4 10-4 10 --4 10 -4
should be interpreted with caution. We have found that O:'-monobutyryl cyclic AMP is rapidly hydrolyzed by CHO cell extracts, while N6-monobutyryl cyclic AMP is slowly hydrolyzed and dibutyryl cyclic AMP is most resistant [8]. This has also been found in other studies [10,11]. For this reason, it is not clear at this time at what rate analogues such as 8-bromo and 8-benzylthio cyclic AMP are hydrolyzed by the phosphodiesterase activity. The ability of the cyclic nucleotide analogues to activate the cyclic AMPdependent protein kinase activity in CHO cell extracts was also studied as another measure of their ability to mimic cyclic AMP action. The protein kinase activity is stimulated 3--5-fold by cyclic AMP and several of the cyclic nucleotide analogues (Fig. 3). The concentration of cyclic nucleotide which is needed for 50% activation is given in Table II. The ability of the butyryl derivatives of cyclic AMP to activate the protein kinase is similar to that found by others [10,19--23]. N6-Monobutyryl cyclic AMP is an effective activator while O2'-monobutyryl cyclic AMP is 10-fold less effective. Dibutyryl cyclic AMP is a poor activator. Both 8-bromo and 8-benzylthio cyclic AMP are very effective activators of the protein kinase, almost identical to cyclic AMP in this respect. Cyclic GMP and its derivatives do not appreciably activate the protein kinase. A comparison of the phosphodiesterase and protein kinase studies reveals an interesting property of the 8-bromo and 8-benzylthio derivatives of cyclic AMP in that this substitution has not appreciably affected the ability to activate the protein kinase. However, inhibition of the cyclic AMP phosphodiesterase has been markedly altered. Based upon these measurements of phosphodiesterase inhibition, all the cyclic AMP derivatives except dibutyryl cyclic AMP appear capable of inhibiting the intracellular cyclic AMP phosphodiesterase activity as proposed for N 6m o n o b u t y r y l cyclic AMP. In dibutyryl cyclic AMP-treated cells, the measured accumulation of N6-monobutyryl cyclic AMP is sufficient to cause significant phosphodiesterase inhibition [7]. However, in the absence of data on intracel-
41
/-/"
z w tO o1 =_
"8 0
i
B O x
5-
3-
- - v
£D ,7 I
0_ (D
0 -B
-5 LOG
NUCLEOTIDE
(M)
Fig. 3. In v i t r o a c t i v a t i o n of cyclic A M P - d e p e n d e n t p r o t e i n kinase b y cyclic n u c l e o t i d e a n a l o g u e s . T h e a c t i v a t i o n of p r o t e i n kinase in C H O cell e x t r a c t s was m e a s u r e d w i t h , in A: ~, cyclic AMP: ~', N 6 - m o n o b u t y r y l cyclic AMP; L7 O 2 L m o n o b u t y r y l cyclic AMP~ m, d i b u t y r y l cyclic AMP: a n d , in B: &, 8 - b r o m o cyclic AMP: A 8 - b e n z y l t h i o cyclic A M P : D cyclic GMP~ i 8 - b r o m o cyclic GMP: ©, N 2 , O 2 L d i b u t y r y l cyclic GMP. Specific a c t i v i t y is e x p r e s s e d as p m o l of [ 3 2 p ] p h o s p h a t e t r a n s f e r r e d t o h i s t o n e p e r rain p e r mg protein.
lular accumulation of the remainder of the cyclic nucleotide analogues, it is impossible to extrapolate in vitro measurements to effects within the cells. Since treatment of cells with dibutyryl cyclic AMP resulted in an increase in intracellular cyclic AMP and the activation of the cyclic AMP-dependent protein kinase [7,9], these two parameters have been studied in cells treated with other analogues. Cultures were incubated with medium containing each of the analogues at a concentration of 1 mM for 6 h. This time interval is sufficient for the morphological conversion to occur. The intracellular level of cyclic TABLE
II
ACTIVATION OF CYCLIC TIDE ANALOGUES
AMP-DEPENDENT
PROTEIN
KINASE
ACTIVITY
BY CYCLIC
NUCLEO-
The ability of cyclic nucleotide analogues to activate cyclic AMP-dependent protein Idnase was measured in cell-free e x t r a c t s by the t r a n s f e r of [ 3 2 p ] p h o s p h a t e f r o m 7-labeled A T P t o h i s t o n e . T h e c o n c e n t r a t i o n w h i c h causes 50% of t h e t o t a l a c t i v a t i o n is p r e s e n t e d ( f r o m Fig. 3). Cyclic n u c l e o t i d e
P r o t e i n k i n a s e a c t i v a t i o n in v i t r o w i t h CHO cell e x t r a c t s ( c o n e . w h i c h causes 50% a c t i v a t i o n , M)
Cyclic AMP N 6, O 2 ' - D i b u t y r y l cyclic AMP N 6 - M o n o b u t y r y l cyclic A M P 0 2 ' - M o n o b u t y r y l cyclic AMP 8 - B r o m o cyclic AMP 8 - B e n z y l t h i o cyclic AMP
1.0 2.0 6.0 7.0 1.3 1.6
Cyclic GMP N 2, O 2 ' - D i b u t y r y l cyclic GMP 8 - B r o m o cyclic GMP
>1 >1 >1
X × X X X X
10 -7 10 -5 10 -7 10 -6 10 -7 10 -7
X 10 -5 X 10 -5 X 10 -5
IN CHO CELLS IN CULTURE
-----+ + + + + + + + -------
Nothing Nothing
--22.1 23.3 7.3 29.8 16.2 31.3 13.4 0.9 1.0 1.3 2.4 0.8 0.9 0.8 0.9 1.0 1.1
Treated (1 r a M , 6 h )
Cyclic AMP level *
* I n t r a c e l l u l a r l e v e l o f c y c l i c i n p m o l / 1 0 6 cells. ** D i f f e r e n c e b e t w e e n t r e a t e d a n d c o n t r o l l e v e l o f i n t r a c e l l u l a r c y c l i c A M P . * * * R a t i o o f p r o t e i n k i n a s e V a c t i v i t y (---cyclic A M P / + c y c l i c A M P ) .
Cyclic AMP O 2 '-Monobutyryl cyclic AMP N6-Monobutyryl cyclic AMP N6-Monobutyryl cyclic AMP N 6, O 2 ' - D i b u t y r y l c y c l i c A M P N 6, O 2 ' - D i b u t y r y l c y c l i c A M P 8-Benzylthio cyclic AMP 8-Benzylthio cyclic AMP 8-Bromo cyclic AMP 8-Bromo cyclic AMP Cyclic GMP Cyclic GMP N 2, O 2 ' - D i b u t y r y l c y c l i c G M P N 2, O 2 ' - D i b u t y r y l c y c l i c G M P 8-Bromo cyclic GMP 8-Bromo cyclic GMP
Cyclic AMP
Cell elongation activity (1 r a M , 6 h )
Cyclic nucleotide
--
-2.4 1.0 1.1 26.4 14.8 28.9 12.5 ---0.2 0.1 0.1 1.3 ---0.1 --0.2 ---0.2 0.1 0.0 0.1
1.1
0.9 19.7 22.3 6.2 3.4 1.4 2.4 0.9 1.1 0.9 1.2 1.1 0.9 1.1 0.9 0.8 1.0 1.0
Control
Net cyclic AMP increase * *
C H O c e l l s i n c u l t t t r e were i n c u b a t e d w i t h e a c h c y c l i c n u c l e o t i d e a t a c o n c e n t r a t i o n o f 1 m M f o r 6 h . C e l l m o r p h o l o g y AMP and the degree of activation of the intracellular cyclic AMP-dependent protein kinase was measured.
ACTIVITY OF CYCLIC NUCLEOTIDES
TABLE III
Control
0.34 0.25 0.38 0.48 0.31 0.36 0.34 0.39 O.3O 0.37 0.31 0.39 0.41 0.32 0.24 0.29 0.36 0.34 0.40
Treated (1 r a M , 6 h ) --0.43 0.52 0.33 0.86 0.99 0.95 1.09 0.95 0.82 0.83 1.01 0.39 0.26 0.32 0.31 0.40 0.33
Protein kinase V activation ratio * * *
was noted and the intracellular level of cyclic
43 AMP and the degree of activation of the cyclic AMP-dependent protein kinase was measured. As described in Materials and Methods, a control was performed for each analogue treatment to ensure that none of the measured effects was due to incomplete washing of the analogue-containing medium from the cultures. For the cyclic AMP measurement the extracts were chromatographed on D o w e x columns to separate the cyclic AMP from the cyclic nucleotide analogue. The cyclic AMP-dependent protein kinase activity in the extracts was measured in the absence and presence of 1 pM cyclic AMP, an a m o u n t sufficient to completely activate the enzyme [9]. Table III summarizes the data on intracellular cyclic AMP level and degree of activation of the protein kinase in the analogue-treated cells. Dibutyryl and N6-monobutyryl cyclic AMP, as well as 8-bromo and 8-benzylthio cyclic AMP cause the morphological conversion while the remainder of the cyclic nucleotides are inactive (Table III, column 1). Cyclic AMP does not affect the cell morphology and appears to cause no change in the intracellular level of cyclic AMP nor activate the cyclic AMPdependent protein. This is consistent with earlier studies which found no accumulation of cyclic AMP intracellularly in cultures incubated with cyclic AMP for up to 24 h [7]. The removal of the medium cyclic AMP from the cultures has posed a problem. However, there is no increase in intracellular cyclic AMP over the control cultures (Table III, columns 2 and 3) nor any activation of the cyclic AMP-dependent protein kinase (columns 5 and 6). Studies with the remainder of the cyclic nucleotides show that all those which induce the morphological conversion also cause the activation of the intracellular cyclic AMP-dependent kinase (Table III, columns 5 and 6). The inactive analogues, O2'-monobutyryl cyclic AMP and the cyclic GMP nucleotides, do n o t induce the morphological change nor activate the protein kinase. However, only in cells treated with dibutyryl and N6-monobutyryl cyclic AMP is there an increase in the intracellular cyclic AMP level (columns 2--5). This increase is presumably a result of the inhibition of cyclic AMP phosphodiesterase activity. There is no change in the cyclic AMP levels in 8-bromo and 8-benzylthio cyclic AMP-treated cultures. This raises the possibility that, although these two analogues are effective phosphodiesterase inhibitors in vitro, they do not accumulate at sufficient intracellular concentrations to inhibit the enzyme within the cells. This may be a result of a low rate of uptake or of hydrolysis by the cyclic AMP phosphodiesterase activity. However, in these treated cells the cyclic AMP-dependent protein kinase is activated. This can be explained by the direct action of the analogues. The in vitro studies of protein kinase activation are consistent with this proposal since both 8-bromo and 8-benzylthio cyclic AMP were as effective as cyclic AMP itself in the activation (Table II). Discussion
The mechanism by which cyclic AMP analogues induce the morphological conversion of CHO cells is n o t fully understood. However, this study implicates cyclic AMP-dependent protein kinase activation in this process. Only those analogues which induce the morphology change cause the in vivo activation of the protein kinase. The mechanism by which the protein kinase is activated
44 appears to differ depending on the cyclic nucleotide analogue used. Two possible mechanisms for cyclic nucleotide analogue action have been discussed, inhibition of cyclic AMP phosphodiesterase which would result in an increase in intracellular cyclic AMP and activation of the cyclic AMP-dependent protein kinase or the direct activation of the kinase by the analogue [7,12]. Dibutyryl and N6-monobutyryl cyclic AMP appear to act through the first mechanism, while 8-bromo and 8-benzylthio cyclic AMP act through the second. The 1 0 30-fold increase in intracellular cyclic AMP which results from treatment with the two butyryl derivatives is sufficient to cause the protein kinase activation, since similar cyclic AMP changes and protein kinase activation are seen in CHO cells treated with prostaglandin E1 or cholera toxin (Li, A.P., O'Neill, J.P. and Hsie, A.W., unpublished). However, there is no change in cyclic AMP level in cells treated with 8-bromo or 8-benzylthio cyclic AMP, and the protein kinase is also activated. It appears that these two analogues activate the intracellular protein kinase directly. It is likely that these two mechanisms of analogue action have general application since these studies of cyclic AMP phosphodiesterase inhibition and cyclic AMP-dependent protein kinase activation in CHO cell extracts have yielded results similar to that found in other studies [24]. A role for protein phosphorylation in the morphological conversion of CHO cells seems likely. This effect differs from other cyclic AMP-mediated effects in that gene transcription is not involved directly since the conversion also occurs in enucleated cells [4]. The involvement of protein kinase activity must then be in the phosphorylation of preexisting cellular components. Microtubule polymerization has been implicated in the morphological conversion and the phosphorylation of tubulin has been reported [6,25--27]. Phosphorylation of tubulin may have a role in microtubule polymerization. Multiple cellular components may be involved since other changes are induced by cyclic nucleotide analogues besides the morphological conversion [1,2]. The distribution of cyclic AMP-dependent protein kinase activity as well as substrate for phosphorylation in several subcellular fractions is consistent with this [28]. In addition, the existence of multiple forms of protein kinase activity in the cytosol of CHO cells, as found in other mammalian tissue [15], allows a variety of phosphorylation reactions to be induced upon treatment with cyclic AMP analogues (Li, A.P. and Hsie, A.W., unpublished).
Acknowledgements We would like to acknowledge the assistance of Dr. Claus SchrSder in the cyclic AMP phosphodiesterase studies, and the helpful advice of Drs. K.B. Jacobson and L.C. Waters in the preparation of the manuscript. The Oak Ridge National Laboratory is operated by the Union Carbide Corporation for the U.S. Energy Research and Development Administration. J.P. O'N. is a postdoctoral investigator supported by a Carcinogenesis Training Grant No. CA05296 from the National Cancer Institute. A.P.L. is a University of Tennessee Predoctoral Fellow.
45 References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Hsie, A.W. and Puck, T.T. (1971) Proc. Natl. Acad. Sci. U.S. 68, 358--361 Hsie, A.W., Jones, C. and Puck, T.T. (1971) Proc. Natl. Acad. Sci. U.S. 68, 1648--1652 Puck, T.T., Waldren, C.A. and Hsie, A.W. (1972) Proc. Natl. Acad. Sci. U.S. 69, 1943--1947 Schr6der, C.H. and Hsie, A.W. (1973) Nat. New Biol. 246, 58--60 O'Neill, J.P., SchrSder, C.H., Riddle, J.C. and Hsie, A.W. (1976) Exp. Cell Res. 97, 213--217 Borman, L.S., Du mont, J.N. and Hsie, A.W. (1975) Exp. Cell Res. 91,422---428 Hsie, A.W., Kawashima, K., O'Neill, J.P. and SchrSder, C.H. (1975) J. Biol. Chem. 250, 984---989 O'Neill, J.P., SchrSder, C.H. and Hsie, A.W. (1975) J. Biol. Chem. 250, 990--995 Li, A.P., Kawashima, K. and Hsie, A.W. (1975) Bioehem. Biophys. Res. Commun. 64, 507--513 Pasternak, Th., Sutherland, E.W. and Henion, W.F. (1962) Biochim. Biophys. Acta 65, 558--560 Falbuard, J.-G., Pasternak, Th. and Sutherland, E.W. (1967) Biochim. Biophys. Acta 148, 99--105 Wagner, K., Roper, M.D., Leichtling, B.H., Wimalasena, J. and Wicks, W. (1975) J. Biol. Chem. 250, 231--239 Kao, F.T. and Puck, T.T. (1968) Proc. Natl. Acad. Sci. U.S. 60, 1275--1281 Brooker, G., Thomas, L.J. and Appleman, M.M. (1968) Biochemistry 7, 4177--4181 Corbin, J.D., Kelly, S.L. and Park, C.R. (1975) J. Biol. Chem. 250, 218--225 Davoren, P.R. and Sutherland, E.W. (1963) J. Biol. Chem. 238, 3 0 0 9 - - 3 0 1 5 Doore, B.J., Bashor, M.M., Spitzer, N., Mawe, R.C. and Saicr, M.H. (1975) J. Biol. Chem. 250, 4 3 7 1 - 4372 Cheung, W.J. (1970) in Advances in Cyclic Nucleotide Research (Greengard, P. and Costa, E., eds.), Vol. 3, PP. 51--65, Raven Press, New York Meyer, R.B. and Miller, J.P. (1974) Life Sci. 14, 1019--1040 Khwaya, T.A., Boswell, Kit., Robbins, R.K. and Miller, J.P. (1975) Biochemistry 14, 4 2 3 8 - - 4 2 4 4 Kaukel, E. and Helz, H. (1972) Biochem. Biophys. Res. Commun. 46, 1011--1018 Kaukel, E., Mundhenk, K. and Helz, H. (1972) Eur. J. Biochem. 27, 197--200 Nedon, F.A. and Bueh, B.M. (1973) J. Biol. Chem. 248, 8 3 6 1 - - 8 3 6 5 Simon, L.N., Shuman, D.A. and Robins, R.K. (1973) in Advances in Cyclic Nucleoside Research (Greengard, P. and Robeson, G.A., eds.), VoL 3, pp. 225--353, Raven Press, New York Grassetti, D.R. and Murray, J.F. (1967) Arch. Biochem. Biophys. 119, 41--49 Easley, C.W. (1965) Biochim. Biophys. Acta 1 0 7 , 3 8 6 - - 3 8 8 Eipper, B.A. (1974) J. Biol. Chem. 249, 1398--1406 Li, A.P. and Hsie, A.W. (1976) Fed. Proc. 35, 1713
Biochimica et Biophysica Acta, 497 ( 1 9 7 7 ) 3 5 - - 4 5 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 28196
ACTION OF CYCLIC NUCLEOTIDE ANALOGUES IN CHINESE HAMSTER OVARY CELLS
J. P A T R I C K O ' N E I L L , A L B E R T P. LI * a n d A B R A H A M W. HSIE
The University of Tennessee, Oak Ridge Graduate School of Biomedical Sciences and the Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 3 7830 (U.S.A.) (Received October 4th, 1976)
Summary Cyclic nucleotide analogues have been tested for their ability to cause the morphological conversion of Chinese hamster ovary cells in culture, as well as for effects on cyclic AMP-related enzymes. The ability of the analogues to inhibit the cyclic AMP phosphodiesterase activity and to activate the cyclic AMP-dependent protein kinase activity in cell extracts has been measured. Cell cultures were incubated with the analogues and the effects on morphology, intracellular level of cyclic AMP, and in vivo protein kinase activation were determined. All analogues which induced the morphological conversion also caused in vivo activation of the cyclic AMP-dependent protein kinase. Only N6,O2'-dibutyryl and N6-monobutyryl cyclic AMP caused on increase in intracellular cyclic AMP, presumably through inhibition of the intracellular cyclic AMP phosphodiesterase activity. The increase in cyclic AMP appears to cause the protein kinase activation. However, analogues such as 8-bromo and 8benzylthio cyclic AMP do not cause any change in intracellular cyclic AMP level and appear to activate the intracellular cyclic AMP-dependent protein kinase directly.
Introduction
Treatment of Chinese hamster ovary (CHO) cells with N6,O2'-dibutyryl adenosine 3' :5'-phosphate (dibutyryl cyclic AMP) causes the usually epitheliallike cells to convert to a fibroblast-like morphology [1--3]. This morphological conversion occurs in enucleated cells [4], shows cell cycle phase specificity [ 5], and involves the reorientation of microtubules [6]. * Present address: Cancer Research and T r e a t m e n t Center, University of N e w Mexico, Albuquerque, N.M., U.S.A.
36 The morphological conversion of CHO cells by dibutyryl cyclic AMP is a rather specific phenomenon. The conversion is not induced by adenine, adenosine, AMP, butyric acid, or unsubstituted cyclic AMP. The inactivity of cyclic AMP can be explained by its rapid degradation by the cyclic AMP phosphodiesterase activity in CHO cells [7]. O2'-Monobutyryl cyclic AMP is also inactive due to its rapid degradation by cyclic AMP phosphodiesterase as well as an O2'-deacylating activity in CHO cells [8]. N6-Mono butyryl cyclic AMP is active, probably due to its relative stability in CHO cells [7,8]. The treatment of actively growing CHO cells with dibutyryl cyclic AMP results in the intracellular accumulation of N6-monobutyryl cyclic AMP and an increase in the endogenous level of intracellular cAMP, presumably due to inhibition of the cyclic AMP phosphodiesterase activity by N6-monobutyryl cyclic AMP [7,8]. This treatment also results in the intracellular activation of the cyclic AMP-dependent protein kinase [9]. We have now extended these studies to other cyclic nucleotide analogues both for effects on properties of cells in culture (in vivo) and on the cyclic AMP-related enzymes in cell-free extracts (in vitro). The ability of these analogues to induce cell elongation, modify the intracellular level of cyclic AMP, and activate the cyclic AMPdependent protein kinase in vivo was investigated. Cell-free extracts were utilized to study the in vitro inhibition of cyclic AMP phosphodiesterase as well as activation of the cyclic AMP-dependent protein kinase. These studies were performed to further define the specificity of the morphological conversion of CHO cells and to attempt to discern the mechanism of action of cyclic nucleotide analogues in this system. The activation of the cyclic AMP-dependent protein kinase has been proposed to be the primary mechanism by which cyclic AMP effects are mediated [10,11]. The mechanism of action of cyclic nucleotide analogues is less clear, although protein kinase activation appears to be involved [9,12]. The analogues may act through inhibition of the cyclic AMP phosphodiesterase to cause an increase in endogenous cyclic AMP, which leads to protein kinase activation, or they may activate the protein kinase directly. In CHO cells the mechanism appears to be dependent on the cyclic nucleotide analogue utilized. Materials and Methods Cell culture. CHO cell clone CHO-K, was employed in these studies and has been described previously [1,13]. The cells were grown in medium F12 supplemented with 10% heat inactivated, dialyzed fetal calf serum (Pacific Biological Co., Richmond, Calif.) in plastic culture dishes under standard cell culture conditions of 5% CO2 in air at 37°C in a 100% humidified incubator. The morphological conversion of the CHO cells by cyclic nucleotides has been described previously [1--3]. Assay of cyclic AMP phosphodiesterase. The measurement of cyclic AMP phosphodiesterase activity in cell extracts has been described in detail previously [7]. The radiodisplacement assay of Brooker et al. [14] was employed, with slight modification, to measure the conversion of 3H-labeled cyclic AMP to adenosine. Several time points were taken with each concentration of sub-
37 strate and inhibitor, usually until 10% of the substrate had been hydrolyzed, to insure that the reaction rate is linear with time. The velocity of the reaction is expressed as pmol of adenosine formed per min per mg of protein. Assay o f cyclic AMP-dependent protein kinase and activation by cyclic nucleotide analogues in vitro. Protein kinase activities were measured by the incorporation of [32P]phosphate from [7-32P]ATP (New England Nuclear, Boston, Mass., > 1 2 Ci/mmol) into calf thymus histone mixture (Aldrich Chem. Co., Milwaukee, Wisc.). The assay developed by Corbin et al. [15] was followed except that 50 mM Tris • HC1 buffer (pH 7.5) was used instead of the 17 mM phosphate buffer (pH 6.8). Assays were performed in the presence or absence of different concentrations of cyclic AMP or other cyclic nucleotides as indicated. The velocity of the reaction is expressed as pmol of [32p]phosphate transferred to histone per min per mg protein. Measurement o f intracellular concentrations o f cyclic AMP. For the measurement of intracellular cyclic AMP, CHO cells were grown to a b o u t 50% confluency on 150-mm plastic culture dishes (Falcon, Oxnard, Calif.). Cultures were incubated with cyclic nucleotide analogues at a concentration of 1 mM in serum-containing media for 6 h. This is sufficient time for the morphological conversion to occur [1]. Since cyclic nucleotide analogues would be expected to interfere with the cyclic AMP-binding assay described below, several precautions were taken to ensure that a measurement of intracellular cyclic AMP was actually being obtained. As controls, plates were treated with medium containing the cyclic nucleotide analogue for approx. I s, that is, the minimum time taken to pour the medium on and off the plates. Both 6-h-treated and control plates were then washed two times with medium, followed by 10 washes with cold 0.15 M NaC1 in 0.01 M potassium phosphate (pH 7.0). These washes were necessary to remove the contaminating traces of media containing the cyclic nucleotide analogue. Untreated cultures were also washed in this manner and only a small decrease in measured steady-state level of cyclic AMP was found. This small effect is probably due to the use of cold saline for the washes which would minimize the energy-dependent efflux of cyclic AMP from the cells [16,17]. Trichloroacetic acid extracts were then made as described previously [7]. Although the medium contamination with cyclic nucleotide analogues was minimized, there was the additional problem of intracellular accumulation of these analogues. Therefore, the extracts were then chromatographed on D o w e x (Sigma Chemical Co., St. Louis, Mo., 50W, hydrogen form) columns to separate cyclic AMP from the analogues as described previously [7]. The elution profile of all the analogues was first determined so that the fractions collected contained only cyclic AMP with minimal contamination by the analogues. The a m o u n t of cyclic AMP was measured by the binding assay described previously [7]. Several dilutions of the extracts were assayed and the total intracellular cyclic AMP is expressed as pmol per 106 cells. Measurement o f the activation o f the cyclic AMP-dependent protein kinase in analogue-treated cells. Cultures were grown and washed exactly as described above for the measurement of intracellular cyclic AMP. The culture dishes were then frozen in liquid nitrogen, thawed in a buffer containing 50 mM Tris • HC1 (pH 7.5), 0.5 M NaC1, 10% glycerol, I mM EDTA, and 13.8 mM fl-mercaptoethanol, and homogenized with a Duall glass homogenizer. After centrifugation
38 at 20 000 X g for 15 min, the supernatant was assayed for protein kinase activity as described above. The assay was performed in the presence and absence of 1 pM cyclic AMP, a concentration which gives m a x i m u m activation of the enzyme and causes dissociation of the holoenzyme into subunits [9]. The ratio of activity in the absence of cyclic AMP to that found in the presence of cyclic AMP is expressed as the protein kinase activity ratio. The activity from untreated cultures is stimulated 3--4-fold by cyclic AMP and has an activation ratio of 0.25--0.35. Completely activated protein kinase would have an activation ratio of 1.0, as reported previously in dibutyryl cyclic AMP-treated cells [7]. Results
We have tested the ability of a number of cyclic nucleotide analogues at a concentration of 1 mM to induce the morphological conversion of CHO cells. We find that dibutyryl cyclic AMP, N6-monobutyryl cyclic AMP, 8-bromo cyclic AMP, and 8-benzylthio cyclic AMP do cause the cells to elongate. Unsubstituted cyclic AMP, O2'-monobutyryl cyclic AMP, cyclic GMP, N2,O2'-dibu tyryl cyclic GMP, and 8-bromo cyclic GMP are inactive in this system. To understand the mechanism by which the active analogues function and the reason for the inactivity of the others, we studied the ability of these analogues to mimic cyclic AMP in two enzymatic reactions (inhibition of cyclic AMP phosphodiesterase and activation of the cyclic AMP-dependent protein kinase). Previously, we reported two phosphodiesterase activities in CHO cells which differ in affinity for cyclic AMP [7,18]. One activity has a high apparent Km for cyclic AMP of 1--3 mM which appears to preclude a physiological role for this activity. Further study has revealed two activities which show low apparent
150-
K i n = 8 . 9 x 10 . 6
I00-
M
~\~
50.
.
o
,b
2'0
v/s
sb
4'0
F i g . 1. E a d i e - H o f s t e e g r a p h o f t h e k i n e t i c s o f d e g r a d a t i o n o f c y c l i c A M P b y c y c l i c A M P p h o s p h o d i e s t e r a s e i n C H O cell e x t r a c t s . A r a n g e o f c y c l i c A M P f r o m 6 X 1 0 - 8 t o 2 X 1 0 - 5 M w a s u s e d . The apparent K m v a l u e s f o r c y c l i c A M P a r e c a l c u l a t e d f r o m the slopes of the l i n e s .
39
0.5-
t/v
0.3.
0.1
10
20
10
20
0.5-
t/v
0.3
0.1
[INHIBITOR] x 10 - 5
Fig. 2. D i x o n g r a p h o f t h e k i n e t i c s o f i n h i b i t i o n o f c y c l i c A M P p h o s p h o d i e s t e r a s e b y N 6 - m o n o b u t y r y l cyclic A M P ( A ) a n d 8-benzylthio c y c l i c A M P (B). C y c l i c A M P w a s p r e s e n t a t 0.3 p M (~), 0 . 5 ~ M (D), 1 . 0 p M (A) a n d 3 . 0 p M (m). T h e a p p a r e n t K i values o f c o m p e t i t i v e i n h i b i t i o n are c a l c u l a t e d f r o m the p o i n t s o f i n t e r c e p t i o n o f t h e lines.
Km values for cyclic AMP (Fig. 1). The ability of the analogues to inhibit the hydrolysis of cyclic AMP in cell-free extracts was measured, and the data obtained with N 6 - m o n o b u t y r y l and 8-benzylthio cyclic AMP are shown in Fig. 2 as typical examples. The inhibition is competitive with respect to cyclic AMP, and two apparent Ki values of inhibition are obtained, presumably reflecting inhibition of both phosphodiesterase activities. The data obtained from these are summarized in Table I. All the cyclic AMP derivatives show two apparent K i values of inhibition. The two m o n o b u t y r y l derivatives and 8-benzylthio cyclic AMP are similar, with 8-bromo and dibutyryl cyclic AMP less effective inhibitors. Cyclic GMP and its t w o derivatives are the least effective inhibitors, and only one apparent K i of inhibition was found. This poor inhibition by cyclic GMP and its derivatives show the cyclic AMP specificity of the phosphodiesterase activities in CHO cells. These inhibition studies show that the cyclic AMP analogues apparently c o m p e t e with cyclic AMP for binding to the phosphodiesterase enzyme. However, the ability of the phosphodiesterase activities to hydrolyze the analogues
40 TABLE
I
INHIBITION LOGUES
OF CYCLIC
AMP
PHOSPHODIESTERASE
ACTIVITY
BY CYCLIC
NUCLEOTIDE
ANA-
The cyclic A M P phosphodiesterase activity in ceU-free extracts of C H O cells was assayed with radiolabeled cyclic A M P as substrate. For cyclic A M P the appaxent K m values are given; for the remainder of the cyclic nucleotides, the appaxent K i values of competitive inhibition are given. The values were calculated from the intercepts of the lines obtained on Dixon graphs. Cyclic nucleotide
Cyclic A M P
Cyclic A M P O2'-Monobutyryl cyclic A M P N 6 - M o n o b u t y r y l cyclic A M P N 6, O2'-Dibutyryl cyclic A M P 8-Benzyltbio cyclic A M P 8 - B r o m o cyclic AMP
1.2 1.1 3.5 2.3 4.0 8.3
cyclic GMP N 2, O 2 ' - D i b u t y r y l cyclic GMP 8 - B r o m o cyclic GMP
1.7 × 10 -3 1.9 X 10 -3 2.0 X 1 0 -3
X X X X × X
10 I0 10 10 10 10
-6 -s -5 -4 -5 -5
pbosphodiesterase activity (M)
8.9 2.5 1.5 7.1 2.1 4.2
× X X × X X
10 -6 10 -4 10 -4 10-4 10 --4 10 -4
should be interpreted with caution. We have found that O:'-monobutyryl cyclic AMP is rapidly hydrolyzed by CHO cell extracts, while N6-monobutyryl cyclic AMP is slowly hydrolyzed and dibutyryl cyclic AMP is most resistant [8]. This has also been found in other studies [10,11]. For this reason, it is not clear at this time at what rate analogues such as 8-bromo and 8-benzylthio cyclic AMP are hydrolyzed by the phosphodiesterase activity. The ability of the cyclic nucleotide analogues to activate the cyclic AMPdependent protein kinase activity in CHO cell extracts was also studied as another measure of their ability to mimic cyclic AMP action. The protein kinase activity is stimulated 3--5-fold by cyclic AMP and several of the cyclic nucleotide analogues (Fig. 3). The concentration of cyclic nucleotide which is needed for 50% activation is given in Table II. The ability of the butyryl derivatives of cyclic AMP to activate the protein kinase is similar to that found by others [10,19--23]. N6-Monobutyryl cyclic AMP is an effective activator while O2'-monobutyryl cyclic AMP is 10-fold less effective. Dibutyryl cyclic AMP is a poor activator. Both 8-bromo and 8-benzylthio cyclic AMP are very effective activators of the protein kinase, almost identical to cyclic AMP in this respect. Cyclic GMP and its derivatives do not appreciably activate the protein kinase. A comparison of the phosphodiesterase and protein kinase studies reveals an interesting property of the 8-bromo and 8-benzylthio derivatives of cyclic AMP in that this substitution has not appreciably affected the ability to activate the protein kinase. However, inhibition of the cyclic AMP phosphodiesterase has been markedly altered. Based upon these measurements of phosphodiesterase inhibition, all the cyclic AMP derivatives except dibutyryl cyclic AMP appear capable of inhibiting the intracellular cyclic AMP phosphodiesterase activity as proposed for N 6m o n o b u t y r y l cyclic AMP. In dibutyryl cyclic AMP-treated cells, the measured accumulation of N6-monobutyryl cyclic AMP is sufficient to cause significant phosphodiesterase inhibition [7]. However, in the absence of data on intracel-
41
/-/"
z w tO o1 =_
"8 0
i
B O x
5-
3-
- - v
£D ,7 I
0_ (D
0 -B
-5 LOG
NUCLEOTIDE
(M)
Fig. 3. In v i t r o a c t i v a t i o n of cyclic A M P - d e p e n d e n t p r o t e i n kinase b y cyclic n u c l e o t i d e a n a l o g u e s . T h e a c t i v a t i o n of p r o t e i n kinase in C H O cell e x t r a c t s was m e a s u r e d w i t h , in A: ~, cyclic AMP: ~', N 6 - m o n o b u t y r y l cyclic AMP; L7 O 2 L m o n o b u t y r y l cyclic AMP~ m, d i b u t y r y l cyclic AMP: a n d , in B: &, 8 - b r o m o cyclic AMP: A 8 - b e n z y l t h i o cyclic A M P : D cyclic GMP~ i 8 - b r o m o cyclic GMP: ©, N 2 , O 2 L d i b u t y r y l cyclic GMP. Specific a c t i v i t y is e x p r e s s e d as p m o l of [ 3 2 p ] p h o s p h a t e t r a n s f e r r e d t o h i s t o n e p e r rain p e r mg protein.
lular accumulation of the remainder of the cyclic nucleotide analogues, it is impossible to extrapolate in vitro measurements to effects within the cells. Since treatment of cells with dibutyryl cyclic AMP resulted in an increase in intracellular cyclic AMP and the activation of the cyclic AMP-dependent protein kinase [7,9], these two parameters have been studied in cells treated with other analogues. Cultures were incubated with medium containing each of the analogues at a concentration of 1 mM for 6 h. This time interval is sufficient for the morphological conversion to occur. The intracellular level of cyclic TABLE
II
ACTIVATION OF CYCLIC TIDE ANALOGUES
AMP-DEPENDENT
PROTEIN
KINASE
ACTIVITY
BY CYCLIC
NUCLEO-
The ability of cyclic nucleotide analogues to activate cyclic AMP-dependent protein Idnase was measured in cell-free e x t r a c t s by the t r a n s f e r of [ 3 2 p ] p h o s p h a t e f r o m 7-labeled A T P t o h i s t o n e . T h e c o n c e n t r a t i o n w h i c h causes 50% of t h e t o t a l a c t i v a t i o n is p r e s e n t e d ( f r o m Fig. 3). Cyclic n u c l e o t i d e
P r o t e i n k i n a s e a c t i v a t i o n in v i t r o w i t h CHO cell e x t r a c t s ( c o n e . w h i c h causes 50% a c t i v a t i o n , M)
Cyclic AMP N 6, O 2 ' - D i b u t y r y l cyclic AMP N 6 - M o n o b u t y r y l cyclic A M P 0 2 ' - M o n o b u t y r y l cyclic AMP 8 - B r o m o cyclic AMP 8 - B e n z y l t h i o cyclic AMP
1.0 2.0 6.0 7.0 1.3 1.6
Cyclic GMP N 2, O 2 ' - D i b u t y r y l cyclic GMP 8 - B r o m o cyclic GMP
>1 >1 >1
X × X X X X
10 -7 10 -5 10 -7 10 -6 10 -7 10 -7
X 10 -5 X 10 -5 X 10 -5
IN CHO CELLS IN CULTURE
-----+ + + + + + + + -------
Nothing Nothing
--22.1 23.3 7.3 29.8 16.2 31.3 13.4 0.9 1.0 1.3 2.4 0.8 0.9 0.8 0.9 1.0 1.1
Treated (1 r a M , 6 h )
Cyclic AMP level *
* I n t r a c e l l u l a r l e v e l o f c y c l i c i n p m o l / 1 0 6 cells. ** D i f f e r e n c e b e t w e e n t r e a t e d a n d c o n t r o l l e v e l o f i n t r a c e l l u l a r c y c l i c A M P . * * * R a t i o o f p r o t e i n k i n a s e V a c t i v i t y (---cyclic A M P / + c y c l i c A M P ) .
Cyclic AMP O 2 '-Monobutyryl cyclic AMP N6-Monobutyryl cyclic AMP N6-Monobutyryl cyclic AMP N 6, O 2 ' - D i b u t y r y l c y c l i c A M P N 6, O 2 ' - D i b u t y r y l c y c l i c A M P 8-Benzylthio cyclic AMP 8-Benzylthio cyclic AMP 8-Bromo cyclic AMP 8-Bromo cyclic AMP Cyclic GMP Cyclic GMP N 2, O 2 ' - D i b u t y r y l c y c l i c G M P N 2, O 2 ' - D i b u t y r y l c y c l i c G M P 8-Bromo cyclic GMP 8-Bromo cyclic GMP
Cyclic AMP
Cell elongation activity (1 r a M , 6 h )
Cyclic nucleotide
--
-2.4 1.0 1.1 26.4 14.8 28.9 12.5 ---0.2 0.1 0.1 1.3 ---0.1 --0.2 ---0.2 0.1 0.0 0.1
1.1
0.9 19.7 22.3 6.2 3.4 1.4 2.4 0.9 1.1 0.9 1.2 1.1 0.9 1.1 0.9 0.8 1.0 1.0
Control
Net cyclic AMP increase * *
C H O c e l l s i n c u l t t t r e were i n c u b a t e d w i t h e a c h c y c l i c n u c l e o t i d e a t a c o n c e n t r a t i o n o f 1 m M f o r 6 h . C e l l m o r p h o l o g y AMP and the degree of activation of the intracellular cyclic AMP-dependent protein kinase was measured.
ACTIVITY OF CYCLIC NUCLEOTIDES
TABLE III
Control
0.34 0.25 0.38 0.48 0.31 0.36 0.34 0.39 O.3O 0.37 0.31 0.39 0.41 0.32 0.24 0.29 0.36 0.34 0.40
Treated (1 r a M , 6 h ) --0.43 0.52 0.33 0.86 0.99 0.95 1.09 0.95 0.82 0.83 1.01 0.39 0.26 0.32 0.31 0.40 0.33
Protein kinase V activation ratio * * *
was noted and the intracellular level of cyclic
43 AMP and the degree of activation of the cyclic AMP-dependent protein kinase was measured. As described in Materials and Methods, a control was performed for each analogue treatment to ensure that none of the measured effects was due to incomplete washing of the analogue-containing medium from the cultures. For the cyclic AMP measurement the extracts were chromatographed on D o w e x columns to separate the cyclic AMP from the cyclic nucleotide analogue. The cyclic AMP-dependent protein kinase activity in the extracts was measured in the absence and presence of 1 pM cyclic AMP, an a m o u n t sufficient to completely activate the enzyme [9]. Table III summarizes the data on intracellular cyclic AMP level and degree of activation of the protein kinase in the analogue-treated cells. Dibutyryl and N6-monobutyryl cyclic AMP, as well as 8-bromo and 8-benzylthio cyclic AMP cause the morphological conversion while the remainder of the cyclic nucleotides are inactive (Table III, column 1). Cyclic AMP does not affect the cell morphology and appears to cause no change in the intracellular level of cyclic AMP nor activate the cyclic AMPdependent protein. This is consistent with earlier studies which found no accumulation of cyclic AMP intracellularly in cultures incubated with cyclic AMP for up to 24 h [7]. The removal of the medium cyclic AMP from the cultures has posed a problem. However, there is no increase in intracellular cyclic AMP over the control cultures (Table III, columns 2 and 3) nor any activation of the cyclic AMP-dependent protein kinase (columns 5 and 6). Studies with the remainder of the cyclic nucleotides show that all those which induce the morphological conversion also cause the activation of the intracellular cyclic AMP-dependent kinase (Table III, columns 5 and 6). The inactive analogues, O2'-monobutyryl cyclic AMP and the cyclic GMP nucleotides, do n o t induce the morphological change nor activate the protein kinase. However, only in cells treated with dibutyryl and N6-monobutyryl cyclic AMP is there an increase in the intracellular cyclic AMP level (columns 2--5). This increase is presumably a result of the inhibition of cyclic AMP phosphodiesterase activity. There is no change in the cyclic AMP levels in 8-bromo and 8-benzylthio cyclic AMP-treated cultures. This raises the possibility that, although these two analogues are effective phosphodiesterase inhibitors in vitro, they do not accumulate at sufficient intracellular concentrations to inhibit the enzyme within the cells. This may be a result of a low rate of uptake or of hydrolysis by the cyclic AMP phosphodiesterase activity. However, in these treated cells the cyclic AMP-dependent protein kinase is activated. This can be explained by the direct action of the analogues. The in vitro studies of protein kinase activation are consistent with this proposal since both 8-bromo and 8-benzylthio cyclic AMP were as effective as cyclic AMP itself in the activation (Table II). Discussion
The mechanism by which cyclic AMP analogues induce the morphological conversion of CHO cells is n o t fully understood. However, this study implicates cyclic AMP-dependent protein kinase activation in this process. Only those analogues which induce the morphology change cause the in vivo activation of the protein kinase. The mechanism by which the protein kinase is activated
44 appears to differ depending on the cyclic nucleotide analogue used. Two possible mechanisms for cyclic nucleotide analogue action have been discussed, inhibition of cyclic AMP phosphodiesterase which would result in an increase in intracellular cyclic AMP and activation of the cyclic AMP-dependent protein kinase or the direct activation of the kinase by the analogue [7,12]. Dibutyryl and N6-monobutyryl cyclic AMP appear to act through the first mechanism, while 8-bromo and 8-benzylthio cyclic AMP act through the second. The 1 0 30-fold increase in intracellular cyclic AMP which results from treatment with the two butyryl derivatives is sufficient to cause the protein kinase activation, since similar cyclic AMP changes and protein kinase activation are seen in CHO cells treated with prostaglandin E1 or cholera toxin (Li, A.P., O'Neill, J.P. and Hsie, A.W., unpublished). However, there is no change in cyclic AMP level in cells treated with 8-bromo or 8-benzylthio cyclic AMP, and the protein kinase is also activated. It appears that these two analogues activate the intracellular protein kinase directly. It is likely that these two mechanisms of analogue action have general application since these studies of cyclic AMP phosphodiesterase inhibition and cyclic AMP-dependent protein kinase activation in CHO cell extracts have yielded results similar to that found in other studies [24]. A role for protein phosphorylation in the morphological conversion of CHO cells seems likely. This effect differs from other cyclic AMP-mediated effects in that gene transcription is not involved directly since the conversion also occurs in enucleated cells [4]. The involvement of protein kinase activity must then be in the phosphorylation of preexisting cellular components. Microtubule polymerization has been implicated in the morphological conversion and the phosphorylation of tubulin has been reported [6,25--27]. Phosphorylation of tubulin may have a role in microtubule polymerization. Multiple cellular components may be involved since other changes are induced by cyclic nucleotide analogues besides the morphological conversion [1,2]. The distribution of cyclic AMP-dependent protein kinase activity as well as substrate for phosphorylation in several subcellular fractions is consistent with this [28]. In addition, the existence of multiple forms of protein kinase activity in the cytosol of CHO cells, as found in other mammalian tissue [15], allows a variety of phosphorylation reactions to be induced upon treatment with cyclic AMP analogues (Li, A.P. and Hsie, A.W., unpublished).
Acknowledgements We would like to acknowledge the assistance of Dr. Claus SchrSder in the cyclic AMP phosphodiesterase studies, and the helpful advice of Drs. K.B. Jacobson and L.C. Waters in the preparation of the manuscript. The Oak Ridge National Laboratory is operated by the Union Carbide Corporation for the U.S. Energy Research and Development Administration. J.P. O'N. is a postdoctoral investigator supported by a Carcinogenesis Training Grant No. CA05296 from the National Cancer Institute. A.P.L. is a University of Tennessee Predoctoral Fellow.
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