5'-Monophosphate-Dependent Protein Kinase Activation and Inhibition Of

(CANCER RESEARCH 43, 3514-3520, August 1983] Kinetics of Adenosine 3':5'-Monophosphate-dependent Protein Kinase

Activation and Inhibition of Thymidine Incorporation into DMA in P1798 Lymphosarcoma Cells1

Carolyn A. Michnoff,2 Becky A. de la Houssaye, and Ruthann A. Masaracchia3

Departments of Chemistry and Biochemistry, North Texas State University, and Texas College of OstÃ©opathieMedicine, DentÃ³n, Texas 76203

ABSTRACT DeWys and Bathina (7) and Mednieks ef al. (19) have applied The kinetics for activation of the cyclic adenosine 3':5'-mon- these observations to the therapy of leukemic lymphocytes. These investigators have observed that elevation of cyclic AMP ophosphate (cyclic AMP)-dependent protein kinase (PKA) and concentrations by methylxanthines dramatically reduced the thymidine incorporation into DNA was investigated in epineph- mortality of mice inoculated with L1210 leukemia cells. The rine- and prostaglandin E, (PGE^-treated murine P1798 lympho- molecular mechanism proposed for this observation includes sarcoma cells. A positive correlation between the duration and increased cyclic AMP accumulation in cells treated with theoph- extent of PKA activation and accumulation of cyclic AMP and ylline and increased activation and intracellular translocation of inhibition of thymidine incorporation into DNA was observed with Isozymes I and II of cyclic AMP-dependent protein kinase both hormones. Epinephrine and PGE, elevated intracellular (ATP:protein phosphotransferase, EC 2.7.1.37). Similar changes cyclic AMP 34- and 14-fold, respectively. All hormone concentra in PKA disassociation and translocation have been reported tions which increased cyclic AMP accumulation also promoted previously in mammary carcinoma cells (4). In contrast to these inhibition of thymidine incorporation into DNA. In addition, dibu- results, Zick ef al, (27) have observed no nuclear translocation tyryl cyclic AMP (50 ^M) inhibited thymidine incorporation. of PKA in activated thymocytes. Furthermore, these studies and No difference in the kinetics for activation of PKA was ob those of Keely ef al. (14) have indicated that apparent translo served when cells were treated with ^M epinephrine or PGE,. cation may be artifactual in some cell systems. Results obtained With both agents, 50% PKA activation was observed when in our laboratory using murine lymphosarcoma cells have sub intracellular cyclic AMP concentrations were elevated 6.5-fold, or to 9 pmol/106 cells. In the presence of Â¿Â¿Mepinephrine,the stantiated the artifactual association of PKA Isozyme I with chromatin and other insoluble cell fractions and have defined cyclic AMP concentration was approximately 3-fold greater than assay procedures which permit analysis of PKA activation cor that required for maximal PKA activation. In this case, the rected for this pseudotranslocation to paniculate fractions (6, duration of the activation time for PKA was also 3- to 4-fold 17,20). longer than that observed with 0.1 MMepinephrine. Since PKA activation and subsequent phosphorylation of The data are consistent with a mechanism wherein both phenotypic substrate proteins are the only mechanisms by which epinephrine and PGE, suppress DNA synthesis by a cyclic AMP- the effects of cyclic AMP are known to be mediated in mammalian mediated cascade of protein phosphorylation. No evidence for cells (16), elucidation of the kinetics of these events is essential independent cyclic AMP or PKA pools which respond independ to understanding the cascade of events involved in the suppres ently to either epinephrine or PGEi could be detected. sion of DNA synthesis in cell populations. This report defines the kinetics of PKA activation in response to 2 agents which increase INTRODUCTION lymphoid cell adenylate cyclase activity and suppress DNA syn thesis. The thymus-derived lymphosarcoma P1798 was selected Prostaglandins of the E series have been shown to increase T-lymphocyte cyclic AMP4 accumulation and to depress the for these studies. Previous studies (6, 17, 18, 24) have shown that this tissue represents a proliferating population of cells which proliferative response to the polyclonal activator phytohaemag- are substantially analogous to other lymphosarcomas (1,13) and glutinin (25). In the past decade since this observation was proliferating lymphoid cells (9-11, 25, 27). reported, several investigators have studied the cyclic AMP mediation of cell proliferation (22). The results of these studies MATERIALS AND METHODS have consistently indicated that inhibition of proliferation in both normal and transformed cells can be affected by cyclic AMP or Cell Preparation. Lymphosarcomas (P1798/R; cortisol resistant) were agents which elevate intracellular cyclic AMP (21, 22). serially transplanted s.c. in 6- to 8-week-old BALB/c mice. Tumors (4 to 6 g) were removed 18 to 21 days after implantation and washed in cold ' Supported by the Robert A. Welch Foundation (Grant B 864) and the American RPMI 1640 (Grand Island Biological Co., Grand Island, N. Y.). All sub OstÃ©opathieAssociation (Grant 80-11-323). sequent steps were carried out at 4Â°.Cells were prepared in RPM11640 2 Present address: Department of Pharmacology, University of Texas Health by the procedure of Rosen ef al. (24). Cell densities were adjusted to Science Center, Dallas, Texas 75234. approximately 1 x 106 cells/ml for experiments in which DNA synthesis 3 To whom requests for reprints should be addressed. 4The abbreviations used are: cyclic AMP, cyclic adenosine 3':5'-monophos- or cyclic AMP synthesis was measured, or approximately 5 x 106 cells/ phate; PKA, cyclic adenosine 3':5'-monophosphate-dependent protein kinase; ml for experiments in which cyclic AMP-dependent protein kinase activity RPMI 1640, Roswell Park Memorial Institute Tissue Culture Medium 1640; PK-1, was measured. For all experiments, cells were incubated at 37Â°for 10 the synthetic peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly; PGE,, prostaglandin E,; PGE2, prostaglandin E2; DBC, /V'.O'-dibutyryl cyclic adenosine 3':5'-monophos- min prior to the addition of test agents. At designated time intervals, phate. aliquots of cell suspensions were withdrawn from the incubation mixture Received August 26,1982; accepted April 21,1983. as described for the individual assay procedures. When present, pros-

3514 CANCER RESEARCH VOL. 43

taglandins were dissolved in 95% ethanol and added directly to the mination with a Glucostat kit. cultures. Final ethanol concentrations did not exceed 0.01% (v/v). This Materials. [-y-32P]ATPwaspreparedby the methodof Walsethand concentration of ethanol did not affect DMA synthesis or cyclic AMP Johnson (26).[/T7e%/-3H]Thymidine(82Ci/mmol)was obtained from ICN accumulation.All other agents were dissolved in 0.15 M NaCIcontaining Chemical and Radioisotope Division (Irvine, Calif.). Prostaglandins, ste 50 mMKH2PO4,pH7.0, or distilled water immediatelyprior to use. roids, and biological amines were obtained from Sigma Chemical Co. cyclic AMP Assay. At designatedtime intervals,duplicate1.0-ml (St. Louis, Mo.). Insulin was obtained from Lilly Pharmaceuticals(Indi aliquots containing approximately 1 x 106cells were withdrawn from the anapolis, Ind.). incubation flasks and pipetted into tubes containing 0.25 ml 40% (w/v) The synthetic peptide' PK-1, which corresponds to the amino acid trichloroacetic acid. Samples were stored at 4Â°until analyses were sequence in pyruvate kinase that is phosphorylated by the cyclic AMP- performed. The protein precipitate was removed from samples by cen- dependent protein kinase (12), was purchased from Sigma. Glucostat trifugation at 4000 x g for 20 min. The supernatant fluid was decanted kits were purchased from Worthington Biochemical Corp. (Freehold, and acidifiedwith 100 ÃŸ\NHCI.The trichloroacetic acid was removed by N. J.). extraction of the aqueous sample with four 3-ml portions of diethylether. Aliquots (50 n\) of the extracted sample were diluted to 500 Â¿ilin5 mw RESULTS sodium acetate, pH 4.5. The samples were acetylated, and the cyclic AMP was determined by the radioimmunoassayof Brooker eÃal.(2). All cyclic AMP Accumulation and Protein Kinase Activation. A assays were performed in duplicate. Antibody to cyclic AMP was kindly dose-dependent increase in cyclic AMP accumulation and PKA provided by Dr. Gary Brooker, Georgetown University, Washington, D. C. The assay reliably measured 10 fmol cyclic AMP. The authenticity of activation was observed when lymphosarcoma cells were incu the measured nucleotide was verified by Dowex 50-X8 chromatography bated with epinephrine (Table 1). No changes in cyclic AMP or by phosphodiesterasetreatment of neutralizedextract. accumulation or PKA activation were observed in cells incubated ProteinPhosphotransferaseAssay.At designatedtimeintervals,1- in the absence of epinephrine. In 7 separate cell preparations, ml aliquots containing approximately 5 x 106cells were withdrawn from the basal cyclic AMP concentrations and PKA activity ratios were the incubation flasks and pipetted into plastic centrifuge tubes in ice. 1.34 Â±0.09 and 0.14 Â±0.06 (S.E.), respectively. On the basis The cell pellets were obtained by centrifugation for 10 sec in an Eppen- of the data in Table 1, the amounts of epinephrine which promote dorf Model 5412 centrifuge. The cell extract was prepared and analyzed half-maximal cyclic AMP accumulation and half-maximal PKA for protein kinaseactivation as described previously(6). Previousstudies (6,17) have established that the principal cyclic AMP-dependentprotein activation were calculated to be 45 and 16 nvi, respectively. Norepinephrine (10 ^M) increased cyclic AMP accumulation kinaseisozymein the lymphosarcomacells is the IsozymeI.The methods established by Keely ef al. (14, 15) for the analyses of cyclic AMP- 3.0-fold and activated PKA approximately 2-fold. Histamine (1 dependent protein kinase activation in intact cells have been adapted for Â¿Â¿M),serotonin(1 /Â¿M),anddopamine (1 /Â¿M)didnot affect cyclic use in the lymphosarcomacells as described in detail elsewhere(6).This AMP accumulation. method of calculation permits correction for catalytic subunit associated Glucocorticoids have been shown to suppress DNA synthesis with paniculate cell fractions and avoids a contribution to the observed in lymphoid cells including the P1798 lymphosarcoma (24); how cyclic AMP-independent enzyme activity by salt-promoted dissociation ever, dexamethasone (1 U.M)did not significantly change cyclic of enzyme and cyclic AMP-independenthistone kinases. Aliquots (20 /Â¿I/ AMP levels or PKA activation, cyclic AMP levels were measured assay)were assayed in duplicate for protein phosphotransferaseactivity. after 10 min as well as after 60-min and 120-min incubations in The phosphotransferase assays were performed by the method of the presence of steroid. None of the cells showed any change in Reiman et al. (23) as adapted by Glass ef al. (&). The aliquots of cell extract were assayed in a total volume of 0.1 ml containing 2 iÂ¡mo\of2- cyclic AMP content. Similarly, protein hormones known to in (N-morpholino)ethanesulfonicacid buffer, pH 6.8, 1 ÃÃmolofMgCI2,and crease cyclic AMP in other cells (glucagon) or to regulate lymph 10 nmolof [7-Å“P]ATP(50to 100 dpm/pmol). Proteinphosphotransferase oid cell metabolism (insulin) did not promote cyclic AMP accu activity of all samples was measured in the absence of added substrate mulation in the lymphosarcoma cells (data not shown). with and without 1 ^M cyclic AMP and in the presence of 200 //M PK-1 The time course for epinephrine stimulation of lymphosarcoma with or without 1 UMcyclic AMP as described previously (6). All incuba cell cyclic AMP accumulation and PKA activation was investi tions were at 30Â°for10 min. The amount of ^P-labeled product isolated gated. When cyclic AMP was determined in cell suspensions was corrected for the phosphorylationof endogenoussubstrates to give incubated 1 to 3 min with 1 ^M epinephrine, maximum cyclic the activity towards the synthetic peptide substrate. Total activity was defined as activity extracted with 0.14 M KCI and measured in the presenceof PK-1 and cyclic AMP. Table 1 Dose dependency of cyclic AMP accumulation and PKA activation in P1798 Thymidine Incorporation Assay. At designated time intervals, 0.2-ml lymphosarcoma cells aliquots containingapproximately 2 x 106cells were withdrawn from the Cells were incubated at 37Â°for 10 min after addition of epinephrine as indicated. incubationflask and pipetted into individualwells of microtiter plates. All Determinations of cyclic AMP concentration and PKA activity were made on determinationswere done in quadruplicate. [3H]Thymidine(0.5 ^Ci) was duplicate cell samples. All determinations are means of duplicate determinations of added to each well, and the cells were transferred to a 37Â°incubator each sample. PKA total activity was 250 pmol Å“P transferred per 1 min per 106 maintained in an air atmosphere. After the 30-min [3H]thymidinepulse, cells. activity ratio the cell suspensions were mechanicallyharvested onto glass fiber filters (- cyclic AMP/+ usinga BrandellModel24-V cell harvester.Incorporationof [3H]thymidine Epinephrine AMP (HM)0 (pmol/106min)1 cells/10 cyclicAMP)0.14 into DNA was determined by liquid scintillationcounting of filter discs in .3 Â±0.06s 5 ml toulene-basedfluor. 10 5.8 Â±0.9 0.26 In all experiments, cell viability was assessed throughout the incuba 20 14 Â±1.6 0.41 tion period. In all reported experiments, cell viability as assessed by 40 24 Â±1.1 0.63 nigrosindye exclusion was 86% or greater at the longest incubationtime 80 40 Â±0.8 0.99 100 52 Â±1.2 0.86 in that experiment. In some experiments, comparative viability of control 200 63 Â±2.7 0.96 and treated cells was assessed by determinations of media glucose 500 55 Â±1.3 0.92 utilization. Cell suspensions were centrifugea at 500 x g for 5 min, and 1000cyclic 56 Â±3.9PKA 1.05 10CM aliquots of the supernatant fraction were used for glucose deter Mean Â±S.E.

AUGUST 1983 3515

AMP accumulation was observed at the earliest time point reassociation of the PKA result from several factors. The insta investigated. After a 1-min incubation with 1 U.Mepinephrine, the bility of epinephrine in aqueous solutions maintained at temper intracellular cyclic AMP levels were 54 Â±2.6 pmol/106 cells as atures and pH utilized in these experiments is well documented. compared to control values of 1.40 Â±0.10 pmol/106 cells in 5 This was directly investigated with the lymphosarcoma cells in cell preparations. In cell suspensions incubated for 3 to 20 min order to establish if the prolonged elevation of cyclic AMP levels with either 1 or 0.1 //M epinephrine, cyclic AMP accumulation observed in the presence of 1 U.Mepinephrine resulted from was maximal after the 3-min time interval (Chart 1). In the continual activation of the adenylate cyclase or slow turnover of presence of 1 UM epinephrine, cyclic AMP levels remained max accumulated cyclic nucleotide. Epinephrine (100 UM) preincu- imally elevated for 10 to 15 min and then declined to approxi bated at 4Â°in either a 0.9% NaCI solution or RPM11640 solution mately 50% of peak level after 20 min. In experiments in which showed no appreciable loss in ability to promote cyclic AMP cells were incubated for prolonged time intervals (20 to 60 min) accumulation during the 60-min time interval investigated. How with 1 UM epinephrine, cyclic AMP accumulation was 1.5 to 3.0- ever, epinephrine preincubated at 37Â°in either 0.9% NaCI solu fold greater than the control value after 60 min, the longest time tion or RPMI 1640 solution lost activity in a time-dependent interval investigated. When the media and cells were analyzed manner (Table 2). The epinephrine dissolved in 0.9% NaCI solu separately for cyclic AMP in these experiments, it was deter tion was inactivated more slowly than the hormone in RPMI mined that essentially all the detectable cyclic AMP was intra 1640. After 60 min of preincubation at 37Â°, however, neither cellular even after a 60-min incubation with 1 UMepinephrine. No solution when added to cell suspensions increased cyclic AMP change in the cyclic AMP levels of control cells was observed in accumulation over control values. Therefore, the decline in intra any of these experiments. cellular cyclic AMP is, in part, due to the inactivation of epineph In the presence of 0.1 UM epinephrine, the cyclic AMP levels rine. began to decline after 3 to 5 min of incubation with agent, and by 15 min, the cyclic AMP values were only 3- to 4-fold greater than those observed in control cells (Chart 1). 1.0 In several experiments, cyclic AMP accumulation and protein o kinase activation were measured after incubation of cell suspen sions with 0.1 to 1 MMepinephrine for time intervals ranging from Å’ 0.8 5 to 60 min. It was consistently noted that, although cyclic AMP t

levels were maximal 5 min after epinephrine addition, protein 06 kinase activity was not maximal at that time point. Data from 2 U 0. representative experiments are shown in Chart 2. In the dose range tested, full activation of the protein kinase was observed lp V 0.4 10 min after epinephrine addition. At higher epinephrine doses, Z i.e., 0.5 to 1.0 fiM, the protein kinase activity remained fully LU activated for 30 to 45 min. At lower doses, i.e., 0.1 Â¿tM,reasso 02 ciation of the enzyme was approximately 50% after 20 min. These observations are consistent with the observed decrease in cyclic AMP accumulation observed in these samples (Chart 5 10 15 20 1). INCUBATION TIME (min) Chart 2. Time course for activation of the cyclic AMP-dependent protein kinase The decrease in cyclic AMP accumulation and concomitant in epinephrine-treated P1798 lymphosarcoma cells. Cells (5x10* cells/ml) were incubated in the presence of 1 UM epinephrine (â€¢),0.1 Â¡Ã•Mepinephrine(A), or no epinephrine {â€¢)at 37Â°for the designated time intervals. Duplicate 1-ml aliquots were withdrawn, and protein kinase activity was determined in the presence (+) and absence (-) of 1 UMcyclic AMP (cAMP). Values are corrected for phosphorylation observed in the absence of PK-1. Activity ratios are calculated as described in "Materials and Methods."

Table 2 Effect of preincubation on epinephrine-mediated cyclic AMP accumulation Epinephrine (100 >IM)in either 0.9% NaCI solution or RPMI 1640 was preincu bated for the designated time intervals at 37Â°.Aliquots (10 pi) were transferred to 1-ml cell suspensions (1 x 106 cells/ml) in RPMI 1640 equilibrated at 37Â°.Control cells were treated with preincubated 0.9% NaCI solution only. Epinephrine, when present, was 1 >IM.After 3 min, a 250-ml aliquot of 40% (w/v) trichloroacetic acid was added to each cell sample, and cyclic AMP was determined as described in "Materials and Methods." Values are means of duplicate determinations. cyclic AMP (pmol/10Â«cells/3 min) 20 30 EpinephrinePreincubation INCUBATION TIME (min) NaCI Chart 1. Cyclic AMP accumulation in P1798 lymphosarcoma cells incubated time (min)05 solution37.5 164040.6 with epinephrine and PGE,. Cells (1 x 106 cells/ml) were incubated in the presence or absence of agent at 37Â°.At the designated time intervals, a 250-/il aliquot of 40% (w/v) trichloroacetic acid was added to each sample, cyclic AMP was deter 1.95 38.2 29.7 mined as described in "Materials and Methods." Control (â€¢);0.1 JIM epinephrine 1020 1.70 40.7 34.0 (A); 1.0 JIMepinephrine (â€¢);10^M PGE, (O); and 5 ^M PGE, (A). Each point is the 1.63 27.4 20.6 mean of duplicate determinations. 30Control1.45 1.600.9% 16.2RPMI 2.84

3516 CANCER RESEARCH VOL. 43

18 - Although differences were observed in the total accumulation of intracellular cyclic AMP in epinephrine-treated and PGEi- treated cells, the time courses for cyclic nucleotide accumulation with the 2 agents were comparable (Chart 1). Maximum cyclic 15 AMP accumulation in response to either 10 or 5 MM PGE, was observed 1 min after agent addition. Similar to the results with epinephrine, the increased cyclic AMP values persisted for a longer time interval with the higher PGEi dose. After 30 min, 12 cyclic AMP values were less than 2.5 times the control value. The increased accumulation of cyclic AMP was specific for E- rf O series prostaglandins. At 1 MM, PGE2 increased cyclic AMP Oo values 2.5- to 3.2-fold. Prostaglandin F2(,and thromboxane B2 00-J ~- Mp did not alter the intracellular cyclic AMP levels (data not shown). The activation kinetics for the PKA in PGEi-treated cells was comparable to that observed with epinephrine. At each PGE, dose tested, the maximum response for that dose was observed by 10 min after agent addition. The subsequent inactivation rate of the protein kinase was dependent on PGE, concentration. In cell suspensions incubated with 5 and 1 MM PGEi, PKA was activated 92 and 51%, respectively. After 30 min, the activity of the cyclic AMP-independent enzyme was 32 and 14%, respec tively. 10"' 1CT6 Since differences in the total cyclic AMP accumulation were observed with epinephrine and PGE,, the possibility was consid [PGE.] ered that the 2 agents generated different intracellular cyclic Charta. Dose response for cyclic AMP accumulation in PGE,-treated cells. AMP pools which did not interact in an identical manner with the Cells (1 x 10s cells/ml) were incubated in the presence of PGE, concentrations as noted. After 10 min, a 250-^1 aliquot of 40% (w/v) trichloroacetic acid was added PKA. Chaplin ef al. (3) have reported evidence which is consistent to each sample. Cyclic AMP was determined as described in "Materials and with functionally distinct pools of cyclic AMP and PKA in lympho Methods." cytes. To test this possibility, the PKA activity ratio and cyclic AMP accumulation were compared in PGE,-and epinephrine- A refractory period in which the lymphosarcoma cells were not treated cells (Chart 4). The correlation between cyclic AMP levels responsive to epinephrine, but retained sensitivity to PGE!, was and PKA activation was identical in epinephrine- and PGEi- observed. The addition of 1 MMepinephrine increased cyclic AMP treated cells, suggesting that no difference in cyclic AMP pools levels from 1.44 pmol/106 cells to 46 pmol/106 cells in 5 min. exists in cells stimulated with these 2 agents. In other experi After 60 min of further incubation, the intracellular content of cyclic AMP was 2.46 pmol/106 in these cells as compared to ments, it was observed that protein kinase activity ratios decline more quickly in PGEi-treated cells as compared to epinephrine- 1.39 pmol/106 in untreated cells. At this time, addition of epi treated cells, presumably as a result of the lowered cyclic AMP nephrine to a final concentration of 1 MM increased cyclic AMP levels in PGE,-treated cells. When complete PKA activation was to 16 and 48 pmol/106 cells in the epinephrine-treated and achieved with either agent, addition of the second agent to the untreated cells, respectively. Addition of 1 MMPGE! to epineph rine-treated or untreated cells resulted in a 14-fold increase in intracellular cyclic AMP in both cases. This is equivalent to a 100 maximum response to PGE, in fresh cells.5 Therefore, the decline < in cyclic AMP was due, in part, to a refractory period which cr follows epinephrine addition to these cells. 80 Comparison of cyclic AMP Accumulation and PKA Activa tion in Epinephrine- and PGEi-treated Cells. Increased intra 60 cellular cyclic AMP accumulation was dose dependent in P1798 05 Ã• cells incubated with PGE, (Chart 3). However, results observed II7 < with PGE, differed from those in Table 1 in 2 aspects, (a) No 40 saturation of the adenylate cyclase response was observed with PGE! concentrations as high as 5 MM,although the lower doses o ce 20 at which increased cyclic AMP accumulation could be detected o. in PGEi-treated cells were comparable to the lower epinephrine doses tested, i.e., 10 to 100 nM. (b) The cyclic AMP accumulation 20 40 60 80 100 observed with comparable doses of epinephrine and PGE, varied CYCLIC AMP (pmol/W cells) by a factor of approximately 3 with epinephrine as the more Chart 4. Cyclic AMP-dependent protein kinase activation in intact P1798 cells. active agent. Cells were incubated at 37Â°for 10 to 30 min in the presence of 20 to 500 nm epinephrine (â€¢)or0.5 to 5 //M PGE, (D)- Aliquota were withdrawn from appropriate 5 C. A. Michnoff, K. S. Harrison, B. A. de la Houssaye, and R. A. Masaracchia. incubation flasks, and cyclic AMP (cAMP) and cyclic AMP-dependent protein kinase activation were determined as described in "Materials and Methods." Data are Suppression of lymphoid cell proliferation by prostaglandins: characterization of the cyclic nucleotide pool and protein kinase activation, submitted for publication. cumulative for 5 different cell preparations.

AUGUST 1983 3517

C.A/vf/c/inor/efa/. cell suspensions did not increase total PKA activity.5 for longer periods of time, and the degree to which thymidine Inhibition of Thymidine Incorporation into DNA by Epineph incorporation was suppressed was increased. However, the rine and PGE,. Inhibitionof thymidine incorporation into DNA in maximum suppression observed in an experiment was 83% at epinephrine-treated cells was observed 60 min after the addition 240 min after addition of 1 UMepinephrine bitartrate. of 1 U.Mepinephrine to the cell suspensions (Chart 5). Suppres The time courses for inhibition of thymidine incorporation by sion of thymidine incorporation increased throughout the 240- epinephrine, PGE,, and DEC were comparable (Table 4). The min incubation time in all 5 cell preparations compared. Similar suppression observed with epinephrine or DBC was comparable; results were obtained with 0.1 U.Mepinephrine. In some experi PGE, was not as inhibitory. This probably reflects the lower ments, thymidine incorporation was measured 30 min after epi cyclic AMP accumulation and shorter PKA activation time ob nephrine addition to cell suspension; i.e., the thymidine pulse served with PGE,. Suppression was also observed with 20 and was begun at the time of epinephrine addition. The epinephrine- 100 U.MDBC. mediated inhibition of thymidine incorporation in these experi In cells incubated with epinephrine and propranolol, the sup ments was quite variable, ranging from 12 to 37% in 4 experi pression response was substantially blocked at longer time ments. However, all epinephrine-treated cell suspensions exhib intervals (Table 4). The suppression observed in short-term ited some inhibition of thymidine incorporation at the 30-min time incubations was variable from experiment to experiment and point. could not be correlated with cyclic AMP accumulation. Since Increased cyclic AMP accumulation, PKA activation, and sup propranolol alone inhibited thymidine incorporation in some ex pression of thymidine incorporation in epinephrine-treated cells periments, this variability may be attributable to a general cyto- were dose dependent (Table 3). At higher doses of epinephrine, toxicity of the drug. This explanation is substantiated by the cyclic AMP accumulation and PKA activation remained elevated observation that cell viability in propranolol-treated cells was decreased by 8 to 14%. Previous experiments (24) have shown that the [3H]thymidine incorporation observed under these conditions reflects thymidine incorporation into DNA and not DNA repair. We have also measured thymidine incorporation into cells incubated with no agents, 1 U.Mepinephrine, 1 U.MPGE,, and 50 U.MDBC. No o differences in [3H]thymidine uptake into these cell suspensions

OC were observed after 30-, 60-, or 120-min incubations (data not o o. er shown). In other experiments, cells were incubated 60 min in the O â€” presence of [3H]thymidine, and then 1 U.Mepinephrine was added is to the medium. After 60 min of additional incubation time, sup et pression of radiolabel incorporation into DNA was observed in g Ã¼ epinephrine-treated cells. The suppression varied from 15 to Z 37%, which was consistent with the results observed when t-I j H Ithymidine and epinephrine were added simultaneously and when cells were harvested 60 min later. The addition of 1.0 or 2.0 /iCi [3H]thymidine during the 30-min pulse increased the total radiolabel incorporated into DNA proportionately, but the degree of suppression observed with 1.0 U.Mepinephrine or 1.0 U.MPGE, 60 120 180 240 was unchanged in these experiments (data not shown). TIME (min) Rosen ef al. (24) and Hallahan ef al. (10) have reported that Chart 5. Time dependence of epinephrine-mediatedsuppression of DNA syn cortisol-mediated inhibition of DNA synthesis in lymphosarcoma thesis in P1798 lymphosarcoma cells. Cells (1 x K^/ml) were incubated at 37Â°in the presence (â€¢)orabsence (O) of 1 MMepinephrine for the designated time intervals. Cells (2 x 10s)were transferred to microtiter wells containing 0.5 nCi Table 4 (3H]thymidine30 min prior to harvest. Cells were harvested, and [3H]DNA was determinedas describedin "Materials and Methods." Points, meanof quadruplicate Suppressionof DNA synthesis by epinephrine, PGÂ£,,anddibutyryl cyclic AMP Cells (1 x 10*) were incubated at 37Â°in the absence of agent (control) or determinations from 5 cell preparations; bars, S.D. presenceof agent as noted. Thirty min prior to harvest, quadruplicate aliquots (200 M!)weretransferred to microtiter wells, and 0.5 MCi[3H]thymidinewas added. Cells were harvested at the designated time intervals as described in "Materials and Tatrfe3 Methods." Values are the percentage of decrease in [3H]thymidine incorporation Comparison of cyclic AMP accumulation, PKA activation, and suppression of DNA as compared to control cells. synthesis in lymphosarcoma cells Cells were incubated as described in Table 1 in the presence of epinephrine DNA synthesis concentrations as noted, and aliquots were withdrawn at 10 min for cyclic AMP (% of suppression) and PKA determinations and at 120 min for [3H]thymidineincorporation determi nations as described in 'Materials and Methods." Epinephrine

activity [Epineph (- cyclic incorpo (5 MM)"0 rine]("M)0 AMP] AMP/+ ration Time (min)0 (1MM)0 (50MM)028 (pmol/10*min)1. cells/5 cyclicAMP)0.14 (cpm)4300 39 Â±0.08a Â±25 60 14 13 14 10 6.58 Â±0.95 0.54 2703 Â±50(38)" 120 36 17 28 44 100[cyclic 43 Â±2.6PKA 0.93[3H]Thymidine1944 Â±24(55) 180 62 2 31 58 " Mean Â±S.E.of quadruplicate determinations. 240Epinephrine(1MM)078propranolol 19PGE, 52DBC 80 " Numbers in parentheses, percentage of suppression. aCell viability in these samples ranged from 80 to 95%.

3518 CANCER RESEARCH VOL. 43

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1983 American Association for Cancer Research. Lymphosarcoma Protein Kinase Activation cells and thymocytes, respectively, involves an inhibition of glu permit evaluation of enzyme activation in intact cells and also cose uptake. Although cortisol did not promote cyclic AMP correct for nonspecific association of subunits with paniculate accumulation in the lymphosarcoma cells, the effect was inves cell fractions (6). Results obtained with these methods show that tigated. No decrease in glucose utilization was observed in the both PGE! and epinephrine activate a common pool of PKA. presence of 1.0 MMepinephrine, 1.0 MMPGE,, or 50 MMDBC as Since no increase in total PKA was observed after intracellular shown in Table 5. In addition, the data provide evidence that cyclic AMP was increased, there does not appear to be meas there was no difference in cell viability at the time intervals studied urable release of PKA from paniculate fractions under these in this report. These data are in agreement with the observation conditions. These results are in contrast to those reported by that no differences in cell viability in control or hormone-treated Chaplin et al. (3). These investigators observed a different pattern cell suspensions analyzed by nigrosin dye exclusion were ob of protein phosphorylation in PGEi- and epinephrine-treated served in any experiments reported herein. lymphocytes activated with concanavalin A. Since results re ported herein provide no direct evidence for discrete pools of soluble PKA, the presence of cyclic nucleotide-independent pro DISCUSSION tein kinases may contribute to the response observed by these In this report, the kinetics of PKA activation has been corre authors. lated with increased cylic AMP accumulation and suppression of Significant suppression of thymidine incorporation into DNA thymidine incorporation in lymphosarcoma cells. Previous studies was observed with partial activation of the protein kinase, al (1, 3, 4, 7, 9, 11, 13, 19, 21, 22, 25) have demonstrated a though the greatest suppression was observed in cells where positive correlation between increased cyclic AMP accumulation cyclic AMP levels and protein kinase activation were maximal. and suppression of lymphoid cell proliferation; however, the The activation of the protein kinase persisted for a relatively cascade of molecular events which establishes a causal relation short time interval as compared to the suppression of thymidine ship between these processes has not been determined. The incorporation which was evident for as long as 4 hr after epi results reported in this study provide further insight into this nephrine, PGE,, or DBC addition to cells. interrelationship by defining the kinetics of the protein kinase The observation that 1 MM epinephrine is more suppressive activation. than 0.1 MM epinephrine provides additional insight into the Although epinephrine and PGEi increased cyclic AMP accu interrelationship between the kinetics of cyclic AMP accumulation mulation to different extents, activation of PKA in response to and PKA activation and the suppressive response. At 0.1 MM changes in intracellular cyclic AMP in cells treated with these epinephrine, maximum cyclic AMP accumulation and PKA acti agents was virtually identical with respect to cyclic AMP concen vation were observed. However, at 1.0 UM epinephrine, the trations and time course of the enzyme activation. Reassociation elevated cyclic AMP values and PKA activation persisted for a of the enzyme subunits was dependent on the cyclic AMP significantly longer time interval. These data indicate that both concentration with epinephrine-treated cells exhibiting a longer the magnitude of the cyclic AMP response and the duration of activation period as a result of the higher intracellular cyclic AMP the response are positively correlated with the suppressive re levels generated by this agent. In contrast to results reported sponse. The data suggest that agents which would prolong the elsewhere (27), no evidence for any separate epinephrine- or cyclic AMP response would increase the suppression response PGE,-generated cyclic nucleotide pools was observed. With each further. agent, the in vivo activation of PKA was accomplished within an The data would support a model in which a suppression of identical cyclic AMP concentration range. Each agent was ca DNA synthesis via a cyclic AMP-dependent mechanism was pable of activating the enzyme completely, and no additional mediated by activation of PKA. In this model, the data support activity was observed when the cells were simultaneously ex the interpretation that the degree of suppression of DNA synthe posed to maximally effective doses of both agents. The evidence sis is proportional to the extent and duration of the PKA activa supports a single cyclic AMP and PKA intracellular pool. tion; however, the data further indicate that simultaneous acti These results are in substantial agreement with those of Zick vation of PKA and maximal suppression of thymidine incorpora ef a/. (27). These investigators showed that translocation of PKA tion do not occur. While the PKA activation is clearly reversible, in thymocytes occurs nonspecifically. In a detailed study of we have not obtained data to indicate that the suppression lymphoid cell PKA activation, we have defined methods which response is reversible. The failure to observe differences in glucose utilization in the treated cells suggests that the response is not cytotoxic in this time interval. Other investigators have Tables suggested that cyclic AMP may arrest cells in the GÃ¬phase of Glucose utilization by P1798 lymphosarcomacells incubated with epinephrine, the cell cycle (21). Our results are not inconsistent with this dibutyryl cyclic AMP. and PGE, Cells (4x10* cells/ml) were incubated in the presence and absence of 1 JIM possibility. epinephrine, 50 JIM DBC, or 1 ,iw PGE, as described in Table 1. Cells were Results in this paper do not directly address the problem of separated from the media by centrifugation at 750 x g for 5 min. Aliquots (100 (d) the intracellular PKA translocation (4, 14, 19, 27), except to were removed from the supernatant fluid for glucose determination as described in "Materials and Methods." establish that the activated PKA is not tightly associated with an medium)Control1.65 (mg/ml insoluble cell fraction and that no change in total protein kinase Incubation time activity is observed after activation by epinephrine, PGEu or (min)0 DBC. It is interesting to note that Mednieks ef a/. (19) have observed translocation of both Isozymes I and II PKA upon 3060 1.62 1.50 1.48 1.48 1.55 activation by increased cyclic AMP in L1210 cells. The P1798 120 1.34 1.28 1.40 1.37 cells, similar to thymocytes (27) and S49 cells (13), contain very 240[Glucose] 0.97Epinephrine1.631.14PGE,1.54 1.00DBC1.65 1.02 little Isozyme II PKA (10% total PKA). Since a cyclic AMP-

AUGUST 1983 3519

a presumed ribonucleicacid. J. Biol. Chem., 248: 2922-2927,1973. mediated suppressive response is clearly observed in the P1798 11. Henry, C. S., Bourne, H. R., and Uchtenstein, L. M. The rote of cyclic 3', 5' cells, the data do not support a functionally distinct role of adenosine monophosphate in the specific cytolytic activity of lymphocytes. J. Isozyme II in this response. Immunol., Ã•08:1526-1534,1972. In summary, the kinetics of PKA activation observed in lymph- 12. Hjelmquist, G., Anderson, J., Edlund, B., and Engstrom, L. Amino acid se quence of a ("P) phosphopeptide from pig liver pyruvate kinase phosphoryl- oid cells treated with epinephrine or PGE, supports a single pool ated by cyclic 3',5'-AMP-stimulated protein kinase and -y-(Å“P)ATP.Biochem. of cyclic AMP and PKA which responds to activation of adenylate Biophys. Res. Commun., 67: 559-563,1974. 13. Hochman, J., Insel, P. A., Bourne, H. R., Coffino, P., and Tomkins, B. M. A cyclase by these agents. The data are consistent with respect structural gene mutation affecting the regulatory subunit of cyclic AMP-de to dose dependency and time course of response with a model pendent protein kinase in mouse lymphoma cells. Proc. Nati. Acad. Sa. U. S. in which suppression of DNA synthesis is promoted by a mech A., 72:5051-5055,1975. 14. Keely, S. L., Corbin, J. C., and Parti, C. R. On the Questions of Translocation anism involving PKA activation. However, a direct correlation of heart cAMP-dependent protein kinase. Proc. Nati. Acad. Sei. U. S. A., 72: between suppression of cell division and the biochemical events 1501-1504,1975. 15. Keely, S., Corbin, J. D., and Park, C. R. Regulation of adenosine 3',5'- described in this paper must await the identification of the monophosphate-dependentprotein kinase. J. Bid. Chem., 250: 4832-4840, phosphoprotein substrates of the cyclic AMP-dependent protein 1975. kinase and the elucidation of the role of these proteins in the 16. Kuo, J. F., and Greengard, P. Cyclic nucleotide-dependentprotein kinases, IV. Widespread occurrence of adenosine 3',5'-monophosphate-dependent pro replication process. tein kinase in various tissues and phyla of the animal kingdom. Proc. Nati. Acad. Sei. U. S. A., 64: 1349-1355,1969. 17. Masaracchia, R., and Walsh, D. A. Protein phosphotransferaseactivities and REFERENCES cyclic nucleotide action in proliferating lymphocytes. Cancer Res., 36: 3227- 3237, 1976. 1. Bourne, H. R., Uchtenstein, L. M., Melmon, K. L, Henney, C. S., Weinstein, 18. McPartland, R. P., Milholland, R. J., and Rosen, R. Nuclear binding of steroid- Y., and Shearer, G. M. Modulation of inflammation and immunity by cyclic receptor complex to lymphosarcoma P1798 resistant and sensitive cells and AMP. Science (Wash. D. C.), 784. 19-28, 1974. effect of concanavalin A on receptor levels. Cancer Res., 37: 4256-4261, 2. Brooker, G., Harper, J., Terasaki, W., and Moylan, R. Radioimmunoassayof 1977. cyclic AMP and cyclic GMP. Adv. 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Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1983 American Association for Cancer Research. Kinetics of Adenosine 3′:5′-Monophosphate-dependent Protein Kinase Activation and Inhibition of Thymidine Incorporation into DNA in P1798 Lymphosarcoma Cells

Carolyn A. Michnoff, Becky A. de la Houssaye and Ruthann A. Masaracchia

Cancer Res 1983;43:3514-3520.

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