Proc. Nat. Acad. Sci. USA Vol. 69, No. 2, pp. 459-462, February 1972 Regulation of Adenosine 3':5'-Cyclic Monophosphate Phosphodiesterase Activity in Fibroblasts by Intracellular Concentrations of Cyclic Adenosine Monophosphate (3T3/dibutyryl cyclic AMP/SV40-transformed cells/Michaelis constants/L cells/prostaglandin El) MASSIMO D'ARMIENTO, GEORGE S. JOHNSON, AND IRA PASTAN Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014 Communicated by Earl W. Sutherland, December 10, 1971 ABSTRACT Cyclic AMP-phosphodiesterase is present also obtained from Schwarz-Mann, snake venom (Ophiophagus in various mouse fibroblasts. Contact-inhibited 3T3 cells hannah) from Sigma Chemical Co., and Dowex 1-X2, 200-400 contain two forms of the enzyme, one with a Km of 2.5 uIM and the second with a Km of 71 AM. As 3T3 cells grow to mesh from Bio-Rad. The resin was washed with 0.5 N NaOH, confluency and cAMP concentrations rise, the activity of 0.5 N HCl, and deionized water, to a final pH of 5.0, before the first enzyme increases, whereas that of the second is use. unchanged. A line of SV40-transformed 3T3 cells with low cAMP concentrations also has low levels of the cAMP- Separatipn and Assay of phosphodiesterase. The cells were phosphodiesterase with a K,,, of 2.5 1AM. Treatment of 3T3 grown under standard conditions (4). The cAMP phosphodi- and SV40-transformed 3T3 cells with dibutyryl cAMP and theophylline increases cAMP-phosphodiesterase accumu- esterase was prepared by washing the monolayer four times lation. This accumulation is blocked by cycloheximide and with ice-cold phosphate-buffered saline (pH 7.4) and four actinomycin D. The newly formed enzyme resembles the times with ice-cold homogenizing medium containing 0.04 M higher affinity enzyme present in unstimulated cells, Tris- HCl (pH 8.0)-10.9% sucrose (w/v). The cells were since it has a Km of 1.2-2.0 MM, and is stimulated by snake scraped from the culture dish with a rubber stopper, and venom. In L cells in which cAMP concentrations are ele- vated by treatment with prostaglandin El, cAMP phos- homogenized in 0.5-1.0 ml of homogenizing medium, at a phodiesterase also accumulates. We conclude that intra- final concentration of protein of 2-5 mg/ml, in a glass Dounce cellular concentrations of cAMP regulate the synthesis of homogenizer (tight pestle; 20 strokes). All steps were at 0- cAMP-phosphodiesterase, and that cAMP functions as an 4°C. The resulting homogenate was used directly as the inducer of the enzyme. enzyme in the assay. Protein measurements were made by the Previous studies indicated that cAMP regulates the growth, method of Lowry et al. (6), with bovine serum albumin as morphology, and adhesiveness of normal and transformed the standard. The assay for cAMP phosphodiesterase was fibroblasts (1-4). Thus, an understanding of cAMP metabo- essentially the two-step assay of Butcher and Sutherland (7), lism is important to elucidate the regulation of these proper- with snake venom used in the second step as a source of ties in normal and cancer cells. 5'-nucleotidase to convert the product of cAMP phospho- cAMP concentrations are determined by the activity of the diesterase (i.e., 5'-AMP) to adenosine and inorganic phos- enzyme adenylate cyclase, which catalyzes the formation of phate. cAMP from ATP, and cAMP phosphodiesterase, which In the first stage, the 0.18-ml reaction mixture contained 40 degrades cAMP into 5'-AMP (for review, see ref. 5). cAMP mM Tris HCl (pH 8), 10 mM MgCl2, 4 mM 2-mercapto- can also leak from cells into the extracellular fluid. We have ethanol, and an appropriate concentration of enzyme. After an previously reported on the properties of adenylate cyclase in equilibration period of 10 min at 300C, during which no loss of normal and transformed fibroblasts (4). Here, we report on the enzyme activity occurred, the reaction was initiated by the properties of the cAMP phosphodiesterase. addition of 20 ul of a solution containing [3H]cAMP (200,000 We find that mouse 3T3 cells have high levels of cAMP cpm) and unlabeled cAMP. After 10 min at 300C, the reaction phosphodiesterase activity; kinetic analysis, suggests the was stopped by heating at 1000C for 1 min. 50 ug of snake activity is due to two enzymes. However, one line of SV4O- venom was added and the components were incubated for a transformed cells has very low activities of cAMP phosphodi- further 10 min at 30°C to convert 5'-AMP to adenosine. This esterase. The total activity of the enzyme is dependent on the reaction was stopped by the addition of 1.0 ml of a 1:3 slurry intracellular concentration of cAMP, and this activity is in- of Bio-Rad AG 1X-2 resin; after they were mixed, the tubes creased by treatment of cells with But2cAMP (N6-2'-O- were centrifuged 10 min at 4000 X g and the radioactivity in dibutyryl cAMP) and theophylline. These findings suggest the supernatant was measured. When snake venom was used that cAMP functions as an inducer of the enzyme. to stimulate the cAMP phosphodiesterase (8), 50 ,g was present in the first stage of the reaction. The blank for each MATERIALS AND METHODS experiment was prepared with heat-inactivated enzyme, or by [3H]cAMP (14.3 Ci/mmole) was obtained from Schwarz- omission of the enzyme from the incubation mixture. The Mann and purified on Dowex AG 50W-X8 resin. cAMP was blank value was subtracted from each experimental value. In all assays the rate of [3H]cAMP hydrolysis was linear for at Abbreviations: But2cAMP, N'-2'-O-dibutyryl cAMP. least 15 min and directly proportional to protein concentration 459 Downloaded by guest on September 27, 2021 460 Biochemistry: d'Armiento et al. Proc. Nat. Acad. Sci. USA 69 (1972) E- .E E c E 8 - B -..0 C .E A S.4 0O-, 4 ~ ~ ~ ~ ~ I-E -0~ ~~~~0 2 1 1.6 32 4 53 8 .026 .040 .064 .OeO .106 .160 I/cAWMPxIOI "M I/cAMPxI M FIG. 2. Kinetic analysis by double-reciprocal plot of cAMP FIG. 1A and B. Kinetic analysis by double-reciprocal plot phosphodiesterase activity in 3T3-SV40 cells. 0-- 0, cAMP of cAMP phosphodiesterase activity in 3T3-4 cells: --- -0, phosphodiesterase activity in the presence of venom: 0-0, cAMP phosphodiesterase activity in the presence of snake cAMP phosphodiesterase activity in the absence of venom. venom: 0-*, cAMP phosphodiesterase activity in the absence of venom. appear to contain two enzymatic activities. One has an ap- parent Km of 2.5,uM and a Vm.x of 200 pmol per mg of protein over a range of 5-250 j&g/ml. In each assay not more than 15- per min (Fig. 1A). The increase of Vm.. due to the presence of 20% of the substrate was consumed. The relative velocity was snake venom in the first step of the reaction is from 200 to 300 expressed in nmol of cAMP hydrolyzed per mg of protein per pmol per mg protein per min. The second enzymatic activity min at 30'C. All of the experiments were repeated from 3 to 5 has an apparent Km of 71 ,uM and a Vma. of 2500 pmol per mg times, and each experimental value represents the mean of two protein per min (Fig. 1B). The presence of snake venom seems or three closely agreeing numbers. to slightly decrease the activity of this second enzyme. RESULTS Table 1 shows the increase in the cAMP phosphodiesterase activity during growth of 3T34 cells. When these cells become Kinetic analysis of the cAMP phosphodiesterase activity in the cAMP phosphodiesterase activity rises Fig. 1A and contact-inhibited, 3T34 cells by double-reciprocal plots is shown in from to 5-fold. This rise is present in both the low- and the B. We find that these cells, like many tissues of the rat (9), 3- high-Km enzymes, and occurs whether the enzyme is measured in the presence or in the absence of venom. The apparent Km TABLE 1. cAMP phosphodiesterase actinty during the growth of 3TS cells TABLE 2. cAMP phosphodiesterase activity in STS-4 cells treated with But2cAMP and theophylline Log-phase Contact-inhibited growing cells cells cAMP phospho- Cyclic AMP (pM) diesterase activity (ref. 3) 3.3 11 (pmol/mg of protein Venom - + - + per min) Vma.. (nmol per Venom mg of protein _+ per min)* 0.1 0.2 0.2 0.3 V.., (nmol per Control 26 48 mg of protein Butc2AMP and theophylline 67 103 per mini)t 1.4 0.6 2.2 2.9 But2cAMP (1.2 mM) and theophylline (1 mM) were added * K., was 2,uM for all four conditions. for 40 hr to a 4-day-old culture. Enzyme activity was measured at t Kin was 71 uM for all four conditions. 0.625 uM cAMP. Downloaded by guest on September 27, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Cyclic AMP-Phosphodiesterase in Fibroblasts 461 values, however, seem to be unchanged. We found that when 3T3-4 cells become contact-inhibited, the intracellular con- centration of cAMP rises by about 3 to 4-fold (3). Therefore, it seems likely that cAMP itself is the inducer of the cAMP phosphodiesterase. To test this possibility, we treated 3T3-4 cells with But2- cAMP and theophylline. The results of these experiments are shown in Table 2. After 40 hr of treatment with 1.2 mM But2cAMP and 1mM theophylline, the cAMP phospho- diesterase activity is more than doubled. We have measured cAMP phosphodiesterase activity in various transformed cell lines.