Activation of Cyclic Nucleotide Phosphodiesterase from Isolated Human Peripheral Blood Lymphocytes by Mitogenic Agenti

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[CANCER RESEARCH 40, 379-386, February1980] 0008-5472/80/0040-0000$02.0O Activation of Cyclic Nucleotide Phosphodiesterase from Isolated Human Peripheral Blood Lymphocytes by Mitogenic Agenti

Paul M. Epstein,2 John S. Mills, Evan M. Hersh, Samuel J. Strada, and W. Joseph Thompson3

Department of Pharmacology, The University of Texas Medical School at Houston (P.M.E., J.S.M., S.J.S., W.J.Tj, The University of Texas System Cancer Center, M. D. Anderson Hospital(E.M.H.), Houston, Texas 77030

ABSTRACT phological alterations resulting in transformation of the lympho cytes from resting to growing cells (9, 20). Although it is not During long-term incubation of human peripheral blood lym known which biochemical events are specific signals for mito phocytes with phytohemagglutinin (PHA), adenosine cyclic 3': genesis, the direct site of action of the mitogens has been 5'-monophosphate (cyclic AMP) phosphodiesterase increases demonstrated to be the lymphocyte cell surface (i 3). Several about 5-fold and guanosine cyclic 3':S'-monophosphate (cyclic lines of evidence have implicated cyclic nucleotides in the GMP) phosphodiesterase increases about i 0-fold in specific modulation or control of mitogen-induced lymphocyte activa activity. Under similar conditions, there is little change in either tion. Rapid increases in cyclic AMP4(32, 46) or cyclic GMP (6, basal or fluoride-stimulated adenylyl cyclase activity. The in 14, i 5) have been reported to occur following treatment of crease in cyclic nucleotide phosphodiesterase activity also human lymphocytes with mitogens. Moreover, lymphocyte mit occurswith other mitogenicactivatorsof blastogenesisinclud ogenesis, under optimal mitogenic conditions, is inhibited by ing concanavalin A, pokeweed mitogen, and Streptolysin 0. cyclic AMP and by agents that increase intracellular concen Cyclic AMP phosphodiesterase activity in PHA-stimulated trations of cyclic AMP (7, i 6, 33, 42). Cyclic AMP also me lymphocytes displays an increase in maximum velocity but no diates the recovery of DNA synthesis in serum-free lymphocyte change in other kinetic properties when compared to that of cultures suppressed by supraoptimal mitogen concentrations unstimulated lymphocytes. Linear density gradient analyses of (4i, 44). cyclic nucleotide phosphodiesterases in mitogen-stimulated Previous studies from this laboratory have shown that iso lymphocytes show an increased activity in lower (3.65) and lated human peripheral blood lymphocytes contain distinct higher (5.9S) molecular weight forms of cyclic AMP and cyclic forms of soluble, high-affinity cyclic nucleotide phosphodies GMP phosphodiesterase. terases (35) and that increased cyclic nucleotide phosphodi Hydroxyurea inhibits PHA-induced mitogenesis but has no esterase activity is associated with growth in Iymphoid cells effect on the observed increase in phosphodiesterase activity. (10). In this report, we describe and analyze an increase in 1-Methyl-3-isobutylxanthmne inhibits PHA-induced mitogenesis cyclic nucleotide phosphodiesterase activity that occurs in and prevents the increase in cyclic GMP phosphodiesterase human peripheral blood lymphocytes following long-term stim activity, but the increase in cyclic AMP phosphodiesterase ulation by mitogens. activity is greater than that seen with PHA alone. The PHA induced increase in cyclic AMP and cyclic GMP phosphodi MATERIALS AND METHODS esterase activity is inhibited by cycloheximide; however, Acti nomycin D does not completely inhibit PHA stimulation of cyclic Isolation of Human Peripheral Blood Lymphocytes nucleotide phosphodiesterase activity when it is used at a concentration that inhibits the PHA-induced increase in [3H]- Human peripheral blood lymphocytes were isolated from 60 thymidine and [3H]uridine incorporation by greater than 90%. to 120 ml of blood from normal donors by defibrination with These results indicate that de novo protein synthesis but not glass beads and separation of the lymphocytes by Ficoll-Hy mitogenesis parse is required for the increased enzyme activity paque density gradient centrifugation (35). Isolated lympho induced by mitogenic agents. The results are discussed with cytes were washed twice with MEM, and the remaining eryth respect to the regulation of cyclic nucleotide phosphodiester rocytes were lysed in hypotonic buffer [280 mm NH4CI-30 mm ase and to the role of these enzymes in lymphocyte prolifera Tris-CI (pH 7.2)] by incubation at 37Â°for i 0 mm. Final cell tion. purity after 2 additional washes in MEM ranged from 80 to 90% lymphocytes, 8 to 20% monocytes, and 0 to 2% granu locytes, as determined by light microscope analysis. INTRODUCTION Mitogenic Stimulation of Lymphocytes Treatment of lymphocytes with nonspecific mitogens such as plant lectins leads to a wide variety of biochemical and mor A. Macroculture Method. Prior to culturing, lymphocytes, isolated as described above, were resuspended in RPMI Me dium 1640 supplemented with i 0% autologous serum; peni

I Supported In part by USPHS Grants GM-21 361 , CA 05831 . and CA 14984 cillin, i 00 units/mI; streptomycin, i 00 @sg/mI;glutamine,0.4 and by the Pulaski County Cancer Society. 2 RecIpient of a fellowship from the Rosalie B. Hite Foundation end a research

starter grant from the Pharmaceutical Manufacturers' Association Foundation. 4 The abbreviations used are: cyclic AMP, adenosine cyclic 3':5'-monophos Present address: Department of Pharmacology, University of Connecticut Health phate; cyclic GMP. guanosine cyclic 3':5'-monophosphate; MEM, minimal es Center, Farmlngton, Conn. 06032. sentiel medium; RPMI, Roswell Park Memorial Institute; BSA, bovine serum 3 To whom requests for reprints should be addressed. albumin; Con A, concanavalin A; PWM, pokeweed mltogen; PHA, phytohemag Received January 16, 1979; accepted November 5, 1979. glutinin; SLO, streptolysin 0; IMBX, 1-methyl-3-lsobutylxanthlne.

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@tmol/ml;andincubated for i hr in 500-mI glass bottles at room phokinase were optimized as an ATP-regenerating system for temperature. Cells were then aliquoted for incubation in a these cells. Although the ATP-regenerating system was nec humidified atmosphere of 5% CO2 in air, as 1 x 10@lympho essary for linear reaction rates for the time and temperature of cytes/mI (10 ml) in 25-sq-cm Falcon No. 301 2 tissue culture incubation, use of a cyclic nucleotide phosphodiesterase inhib flasks (37Â°)or as 0.33 x 1O@lymphocytes/mI (3 ml) in 16- x itor did not affect the production or preservation of the reaction 125-mm Falcon No. 3033 or glass tissue culture tubes. Mito product formed in the presence of the cyclic AMP â€œtrappingâ€• gens or solvent controls were added as indicated. system. B. Microculture Method. Conditionswerethe sameas those used for macroculture except that lymphocyte cells were dis Assay of Cyclic hosphodiesterase Activity pensed with a sterile Hamilton gastight syringe into Falcon No. 3040 Micro-Test II tissue culture plates as 1.5 x 1O@lympho Cyclic nucleotide phosphodiesterase activity was measured cytes/well. as described previously (10). MEM-washed cell pellets were suspended in 40 mm Tris-Cl (pH 8.0)-S mm 2-mercaptoethanol Measurement of [3H]Leucine, (3H]Urldine, and [3H]Thymi and sonically disrupted for 10 sec at setting 50 (80 watts) with dine Incorporation the microprobe of a Biosonik IV sonicator. Microscopic exam ination showed >99% cell breakage. A. Macroculture. Five @sCiof[3H]thymidine(specificactivity, Unless otherwise indicated, reaction mixtures (0.4 ml) con 1.9 Ci/mmol) were added to lymphocytes in macroculture 3 hr tamed 40 mm Tris-CI (pH 8.0), 10 mm MgCI2,5 mm 2-mercap before the termination of cell incubation. Cell incubation was toethanol, cyclic [3HIAMP (0.25 @m, @100,000cpm) or cyclic terminated by cooling cells to 4Â°,washing with 0.9% NaCI E3HIGMP(0.1 (Un, @100,000cpm), and 0 to 0.2 ml (0 to 2 x solution (4Â°),precipitating with 5% trichloroacetic acid (30 mm, 10@cellequivalents) of cell sonicate. Reactions were incubated 40), and washing an additional time with trichloroacetic acid at 30Â°,usually for 10, 20, and 30 mm, and terminated by and then with methanol. Acid-precipitable material was dis boiling for 1 mm. After cooling, 0.1 ml of snake (Ophiophagus solved in 0.5 ml of Soluene (30 mm, 60Â°),and radioactivity hannah) venom (0.5 mg/mI) was added, and the samples were was measured in 10 ml of PPO-POPOP in toluene scintillation incubated for 10 mm at 30Â°.One ml of methanol was added to fluid. each sample and [3Hladenosine or [3Hlguanosine was sepa B. Microculture. One @Ciof[3H]thymidmne(specificactivity, rated from unreacted cyclic E3HIAMPor cyclic (3H]GMP by 1.9 Ci/mmol), [3H]uridine (specific activity, 28 Ci/mmol), or chromatography on Oowex 1-X8 anion-exchange resin. Under [3H]Ieucmne(specific activity, 15 Ci/mmol) was added to each these conditions, recovery of [3H]adenosine and (3H]guanosine microwell 8 hr before termination of the cell incubation. Cells was 80% and blank values were @1.5%.One unit of activity were collected on glass fiber filters and washed 15 times with is defined as the formation of 1 pmol of [3Hjguanosine or 0.9% NaCI solution using a Multiple Automatic Sample Har [3Hjadenosine per mm. Assay linearity was verified from 0 to vester (37). Filters were dried, and radioactivity was determined 30 mm and from 0 to 2 x 106 cell equivalents. using liquid scintillation techniques. Incorporation of all radio Protein was determined either by the method of Lowry at a!. activity was linear for 8 hr. (21) or by the fluorescent method of Udenfriend et a!. (39). DNA was determined by measuring enhancement of ethidium Assay of Adenylyl Cyclase Activity bromide fluorescence as described by Karsten and Wollenber Lymphocytes in culture were pelleted and suspended at ger (17). 101/ml in 0.32 m sucrose-50 mm Tris-Cl(pH 7.4)and sonicated Sucrose Density Gradient Centrifugation in 0.45-mI volumes for 10 sec with the microprobe of a Biosonik IV sonicator (Bronwill Scientific, Rochester, N.Y.) at a setting Five-mi linear (5 to 20%) sucrose gradients containing 40 of S (-â€˜.â€˜5watts).As shown for other cell systems (2), these cells mm Tris-CI (pH 8.0), 5 mm 2-mercaptoethanol, and 10 mm appear intact by light microscope examination and have activ MgCI2 were prepared with a Buchler density gradient mixer. ities which are greater than those obtained by homogenization Cell pellets containing 10 to 75 x 106 cells were sonicated in of the cells. 0.2 ml of the same buffer and layered on the gradients. Three Reaction mixtures (0.25 ml) contained 50 mm Tris-CI (pH mg of BSA, layered on a separate gradient, were used as a 7.4); phosphocreatine, 0.4 mg/mI; creatine phosphokinase, standard. Gradients were centrifuged for I 5 to 16 h (4Â°)at 0.2 mg/mI; 4 mm MgCI2;0.8 mm cyclic AMP; BSA, 0.6 mg/mI; 192,000 x g@ in Beckman SW 50.1 rotors. Fractions were [a-32P@ATP(20 @mi;1 x 10@cpm); 1 x 10Â°sonicated cells; collected using a Buchler density gradient piercing unit (4Â°). and, where indicated, 10 mm NaF. Reactions were incubated The BSA peak was determined by absorbance measurement for 15 mm at 30Â°and terminated by the addition of 50 @tlof1 at 280 nm. Aliquots (10 to 140 @slofeach fraction) were m acetic acid with vigorous stirring. CycIic[3H@AMP(9000cpm) assayed for cyclic AMP and cyclic GMP phosphodiesterase was added (0.7 ml) to estimate recovery (@80%), and the activity to obtain activity profiles. A value of 4.35 was assigned reaction mixtures were adsorbed on 0.5- x 8-cm columns of to BSA for calculation of other sedimentation coefficients. alumina (bottom, 6 cm) and MnO2(top, 2 cm). Cyclic [32P]AMP and cyclic [3H@AMPwereeluted with 1.6 ml of 50 mm Tris-CI Materials (pH 7.4). Radioactivity in the total column eluate was deter mined by liquid scintillation techniques. One unit of activity is Cyclic [8-3H]AMP (specific activity, 25 Ci/mmol), cyclic [G defined as the formation of 1 pmol of cyclic [32P]AMPper mm 3H]GMP (specific activity, 8.4 Ci/mmol), [methy!-3H]thymidmne from [32P]ATP. (specific activity, 1.9 Ci/mmol), and [5-3H]uridine (specific The concentrations of phosphocreatine and creatine phos activity, 23 Ci/mmol) were purchased from Schwarz/Mann,

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Orangeburg, N.Y. Cyclic [3H]AMP and cyclic [3H]GMP were purified by Dowex 1-X8 (200 to 400 mesh) anion-exchange chromatography and stored at â€”20Â°inacidic 50% ethanol. Snake (Ophiophagus hannah) venom, cyclic AMP, cyclic GMP, RNase, and pronase were from Sigma Chemical Co., St. Louis, Mo.; 2,7-diamino-1 0-ethyl-9-phenylphenanthridium bromide (ethidium bromide), Actinomycin 0, and cycloheximide were from Calbiochem, San Diego, Calif.; Ficoll and Con A were from Pharmacia Fine Chemicals, Inc., Piscataway, N. J.; Dowex 1-X8 (200 to 400 mesh) was from Bio-Rad Laboratories, Rich mond, Calif.; Hypaque-M, 90%, was from Winthrop Laborato ries, New York, N.Y.; enzyme grade sucrose was from Merck and Co., Inc., Rahway, N.J.; RPMI Medium 1640, MEM, 1- glutamine, penicillin-streptomycin solution, and PWM were from Grand Island Biological Co., Grand Island, N.Y.; glass beads(3 mm diameter) were from Scientific Products, Houston, Texas; 4-phenylspiro[furan-2(3H), 1â€˜-phthalan]-3,3'-dione (Fluram) was from Roche Diagnostics, Nutley, N.J.; Soluene was from Packard Instrument Co., Downers Grove, Ill.; PHA (PHA-M), and SLO were from Difco Laboratories,Inc., Detroit, Mich. PHA was weighed, dissolved in RPMI Medium 1640, as 10 mg/mI, filter sterilized, and stored frozen for use. PWM and SLO were rehydratedwith RPMI mediumaccording to manu facturer's specifications and stored frozen. Other purchased chemicals were of commercial analytical grade quality.

RESULTS

Stimulation of Lymphocyte Phosphodiesterase Activity by Mitogens. Mitogenic stimulation of human peripheral blood lymphocytes by PHA resulted in 5- to 10-fold increases in the specific activities of cyclic AMP and cyclic GMP phosphodies terases. Cyclic nucleotide phosphodiesterase activity in PHA treated lymphocytes began to increase only after 24 hr in culture and was near maximum at about 3 days, which corre sponded to the time of maximum [3H]thymidmneincorporation (Chart 1, A and C). Long-term increases in lymphocyte phos phodiesterases were consistently observed in separate exper iments, although the magnitude of the increase varied. Adenylyl cyclase activity fluctuated during the culture period, but no activation in PHA-treated cultures was apparent (Chart I B). Both control and PHA-treated lymphocytes showed a 2- to 3-fold decrease in activity in basal and fluoride-stimulated 0 1 2 3 4 5 6 adenylyl cyclase activity after 24 hr in culture. Partial restora DAYSIN CULTURE tion of activity occurred in PHA-treated cultures at 3 days, but Chart 1. Time course of phosphodiesterase activity, adenylyl cyclase activity, and thymidine incorporation in PHA-stimulated and resting human peripheral the specific activity still remained below the zero time value. blood lymphocytes. Lymphocytes from the same donor, cultured at the same Kinetic and Physical Analysis of Increased Phosphodies time, were used for all assays. Culturing was by the microculture method with terase Activity. Comparison of the kinetics of cyclic AMP PHA-M, 100 @zg/ml.cAMP, cyclic AMP; cGMP, cyclic GMP. phosphodiesterase in PHA-activated and resting peripheral blood lymphocytes indicated an increase in the VmaxOf the change in the kinetic parameters of the enzyme system or to PHA-treated cells with no change in the Km(Chart 2). the appearance of a new enzyme form, but rather to a general The increased phosphodiesterase activity in 3-day PHA-stim increase in the activation of the same enzyme forms which are ulated lymphocytes was further examined by linear sucrose already present in the cell. density gradient centrifugation. A general increase in both the Webb et a!. (43) demonstrated that lymphocytes can be high (5.98)- and low (3.65)-molecular-weight forms of cyclic activated by lectins in the absence of serum. Since serum by AMP and cyclic GMP phosphodiesterasewas observed, as itself can influence phosphodiesterase in other cell systems compared to control cells cultured for 3 days in the absence of (26â€”28),PHAwas tested for its ability to stimulate lymphocyte PHA (Chart 3). An increase in both molecularweight forms of cyclic AMP phosphodiesterase in the absence of serum. A 4- cyclic AMP phosphodiesterase was also seen in cells stimu fold increase in both forms of cyclic AMP phosphodiesterase lated by Con A, PWM, and SLO (data not shown). These data occurred in the absence of serum, as compared to control cells suggest that the increase in enzyme activity is due not to a not treated with PHA (data not shown). Although this increase

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1980 American Association for Cancer Research. P. M. Epstein et a!. was less than that observed in the presence of serum, it optimal at the same doses of PHA which produced an optimal nevertheless indicates that phosphodiesterase activity can be mitogenic response (Chart 4). activated by lectins even in the absence of serum. Both high Four different mitogens (PHA, Con A, PWM, and SLO) were (330 @zg/mI)and low (33 @sg/mI)dosesof PHA appeared to tested for their effect on cyclic nucleotide phosphodiesterase stimulate each form of cyclic AMP phosphodiesterase to the activities, and increases in both cyclic AMP and cyclic GMP same extent (data not shown), indicating that at either dose of phosphodiesterase activities were seen for all 4 mitogens (Ta PHA a similar type of activation occurs. ble 1). The increase in cyclic AMP phosphodiesterase specific Relationship of Increased Phosphodiesterase Activity to activity ranged from 7- to 10-fold, and the increase in cyclic Mitogenesis. Temporally, the sharp increase in phosphodies GMP phosphodiesterasespecificactivityrangedfrom5- to I 3- terase activity occurred at about the time of the onset of fold. Although increased phosphodiesterase activity occurred mitogenesis, as indicated by the enhanced rate of DNA synthe in response to each mitogen, the magnitude of the increases sis (Chart 1). The relationship between the increase in phos did not closely correlate with the degree of stimulation of phodiesterase activity and DNAsynthesis was further examined [3H]thymidine incorporation. Insulin (10@ m) produced no de by comparing the effect of different doses of PHA on these 2 tectable increase in phosphodiesterase activity or [3H]thymi processes. At varying doses of PHA, increased cyclic nucleo dine incorporation and had no effect on PHA activation of tide phosphodiesterase activity appeared to correlate with in lymphocytes when added in culture with PHA for 3 days. creased DNA synthesis; the increase in phosphodiesterase The relationship between phosphodiesterase and mitogene activity was dependent on the dose of PHA used and was sis was further tested by using specific enzyme inhibitors. We found that IMBX is a potent inhibitor of lymphocyte cyclic AMP @ phosphodiesterase (K 6 @tm).When1 mm IMBX was added Km =O.4@iM with PHA at the start of lymphocyte activation, it completely vm =O.S5pmol/mln/1O'c.lls inhibited DNA synthesis and the increase in cyclic GMP phos phodiesterase activity (Table 2); however, the increase of cyclic AMP phosphodiesterasewasstillobserved.At 0.1 mm IMBX, DNA synthesis was inhibited by about 50%, and the increase in cyclic GMP phosphodiesterase was inhibited by about 35%, but cyclic AMP phosphodiesterase activity increased to an even greater extent with the addition of IMBX than with PHA alone (Table 2). In contrast to PHA, which increases the activity of both forms of cyclic AMP and cyclic GMP phosphodiester ase, as analyzed by sucrose gradient fractionation (see above), we found that incubation of lymphocytes with IMBX alone specifically leads to a long-term increase in the lower-molecu lar-weight (3.65) form of cyclic AMP phosphodiesterase, with no change in cyclic GMP phosphodiesterase (34). When IMBX 15 and PHA are added in culture together, they appear to be

CAMP (iaM) synergistic in their activation of the lower-molecular-weight Chart 2. Representative Lineweaver-Burk (19) plots of cyclic AMP phospho form of cyclic AMP phosphodiesterase (data not shown). diesterase activity in human peripheral blood lymphocytes cultured for 72 h by Effect of Inhibitors of Macromolecular Synthesis on Mito macroculture, with or without PHA, 100 pg/mI. Enzyme activity was assayed as gen Stimulation of Phosphodiesterase Activity. When hy @ described in Materials and Methodsâ€•atsubstrate concentrations from 0.05 to 50 @M,using0.5 x 106 cell sonicate equivalents per assay for control cells and droxyurea, a specific inhibitor of deoxyribonucleotide reduc 0.25 x 1o@cellsonicate equivalents per assay for PHA-stimulatedcells. Apparent tase (38), was added with PHA to lymphocyte cultures at Michaelis constants (Km) and apparent maximum velocities [vma, (Vm)] were determined from the linear portions of the plots according to the method of concentrations that inhibited DNA synthesis (Table 2), activa Cleland (5). tion of cyclic AMP and cyclic GMP phosphodiesterase still

Chart 3. Linear sucrose gradient fractionation of cyclic I- AMP phosphodiesteraseandcyclicGMP phosphodiesterase U in human peripheral blood lymphocytes cultured for 72 hr in macroculture either with or without PHA-M, 100 @g/ml.Thirty x 106 PHA-stimulated and 75 x 106 control cell equivalent 10,000 x g supernatant was applied to each gradient, but for graphical clarity results were normalized to 30 x 10Â°cellsfor both PHA and control gradients. Enzymeactivity was essayed as described in â€œMaterialsandMethodsâ€•exceptthat sub strete concentrations used were 0.025 @uncyclicAMP and r 0.1 @tmcyclicGMP.

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occurred. Hence, activation of phosphodiesterase activities lecular weight, kinetic properties, and affinity for substrate (1). can occur under conditions where mitogenesis is completely The functional significance and interrelationship between these inhibited. different forms is not well understood. One means of under Other inhibitors of macromolecular synthesis were examined standing this enzyme system better is to study the factors and for their effects on PHA activation of lymphocyte phosphodi conditions which regulate changes in its activities. Evidence esterase. Cycloheximide at 2 and 20 gsg/mI completely in has accumulated to indicate that many mammalian phospho hibited PHA stimulation of both cyclic AMP and cyclic GMP diesterases, mostly membrane-bound forms, are under hor phosphodiesterase activity and [3H]thymidmneincorporation monal control (36). We have previously demonstrated the ex (Table 2). At 0.2 @g/ml,cycloheximide inhibited [3H]thymidine istence of high-affinity forms of soluble phosphodiesterases in incorporation by 93% and inhibited the increase in both cyclic human peripheral blood lymphocytes (10, 35). In this paper, AMP and cyclic GMP phosphodiesteraseactivities by 90%. we demonstrate severalfold activation of both cyclic AMP and This suggests that the PHA-induced increase in phosphodies cyclic GMP phosphodiesterase in lymphocytes following treat terase activity is dependent on de novo protein synthesis. ment of the cells with mitogens. Actinomycin D at low concentrations (0.5 to 50 ng/ml) was Lectins have been shown previously to affect enzymes in also tested for its effects on macromolecular synthesis and volved in cyclic nucleotide metabolism. PHA has been reported phosphodiesterase activation in response to PHA (Chart 5). As to activate human lymphocyte adenylyl cyclase in vitro (32) shown previously by others (18), at 50 ng/ml, actinomycin 0 and to decrease murine thymocyte adenylyl cyclase when completed inhibited [3H)uridine and [3H]thymidine incorporation added to intact cells (22). Activation of membrane-bound cyclic in PHA-stimulated cultures. At this concentration, actinomycin AMP phosphodiesterase has been described in the cellular D also completelyinhibitedthe PHA-inducedincrease in both slime mold, Dictyoste!ium discoideum (12), and in skeletal cyclic AMP and cyclic GMP phosphodiesterase. However, at muscle (8) following treatment with Con A. Monahan et a!. (23) concenfrations below 50 ng/ml, the inhibition of [3H]uridmne reported that cyclic AMP phosphodiesterase activity in lympho and (@H]thymidmneincorporationby actinomycin 0 was more cytes from normal donors and from chronic lymphocytic Ieu pronounced than the inhibition of phosphodiesterase activation kemia patients is increased approximately 2-fold at 3 to 5 days (Chart 5, inset). following treatment with PHA. These results confirm and extend the observation of Monahan et a!. and provide the first full DISCUSSION description of activation of mammalian cyclic nucleotide phos phodiesterases by plant lectins; hence, these agents must be Most mammalian cells appear to contain multiple molecular added to the growing list of effectors of mammalian phospho forms of cyclic nucleotide phosphodiesterase differing in mo diesterases.

a' -I E I C E 0 .5 0 z m Q. 20

>. z I-. 0 C.) 0 > a. I- 15 0 0 C) > â€˜U (1) -I I- 0 C z Cl). LU0 @20 0 0 a.I (4) I 0 I I 10 100 1000 a. PHA (pg/mI) I Chart 4. Dose response of PHA on cyclic nucleotide phosphodlesterase activity and on [3Hjthymidine incorporation. Cells were cultured by microculture, end @ essays were performed as described In â€˜MaterialsandMethods. â€˜Resultsrepresent the average of duplicate determinations of enzyme activity and quadruplicate determinations of thymidine incorporation. cAMP, cyclic AMP; cGMP, cyclic GMP.

FEBRUARY1980 383

Table 1 lymphocytesCellsIncrease in cyclic nucleotide phosphodiesterase activity following mitogen stimulation of human peripheral blood inâ€˜Materialswere cultured for 7 days by the macroculture method In I 6- x I 25-mm glass culture tubes, at 1 @6lymphocytes/tube,as described 1:30dilution.and Methods.â€•Concentrationsof mitogenic agents used were: PHA, 330 gig/mI; Con A, 27 pg/mI; PWM, 1:30 dilution; SLO, All results represent the average of duplicate determinations.Cyclic nucleotide phosphodlesterase activity

Cyclic AMP (0.25 ELM) Cyclic GMP (0.1 @M) [3HJlhymidlne in corporationAgent Protein Units/mg pro- Units/mg pro- (cpm)Control (@Lg) Total units tein Total units tein 1.700PHA 72 0.18 2 0.01 7 0.2 135,000ConA 145 2.5 17 0.38 2.6 54.000PWM 170 2.6 15 0.20 1.2 157,000SLO 162 3.0 19 0.22 1.4 132 1.9 14 0.14 1.1 146.000

Table 2 Effect of specific enzyme inhibitors on PHA activation of cyclic nucleotide phosphodiesterases asdescribedCells were cultured for 72 hr In microculture. Enzyme activity was essayed aspmol/mln4tgin â€œMaterialsendMethods,â€•endspecific activity was determined controlvalue. DNA. Stimulation index is the ratio of experimental value to 0.190pmol/mln/@gActual control values were: cyclic AMP phosphodiesterase activity, DNA; cyclic GMP phosphodiesterase ectivity, 0.030 pmol/min4ig DNA;[3H]thymldlneincorporation,220 cpm;totalcells.Stimulation DNA, 1.2 @&g/106 indexPhosphodiesterese

spe cific activity

CyclicTotal(0.25 AMP cyclic GMP [3HjThymidine DNAControl @M) (0. 1 @tM) incorporation 1PHA 1 1 1.0 3.8PHA(100 @g/ml) 4.6 219 4.0 3.3hydroxyureaPHA+ 0.5 mM 5.7 15 1.5

3.9PHA+ 1 mMIMBX 0.8 1 1.5 6.1PHA+ 0.1 mM IMBX 3.0 103 2.1 0.2@g/ml)PHA+ cycloheximide (20 0.3 0.4 1.4

0.3@g/ml)PHA+ cycloheximide (2 0.3 0.6 1.6

1.3(0.2+ cycloheximide 1.4 16 2. 1 @tg/ml)

These results show that mitogenesis per se is not required for lectin-induced phosphodiesterase since hydroxyurea, IMBX, and actinomycin 0 all inhibited mitogenesiswithout ACTINOMYCIN D completely inhibiting the activation of phosphodiesterase activ Chart 5. Effect of ectinomycin D on PHA-stimulated Increases In cyclic nu ity. Therefore, the 2 processes appear not to be interdepend cleotide phosphodlesterase activities and In rates of mecromolecular synthesis. ent. However, activation of phosphodiesterase could be a Cells were cultured by the macroculture method with PHA, 100 g@g/ml.Inset: necessary step involved in the actions of plant lectins. Activa abscissa, % of inhibition of PHA stimulation of cyclic AMP phosphodlesterese determined at 68 hr; ordinate, % of Inhibition of PHA stimulation of [3H]urldlne tion of phosphodiesterase was seen only at lectin concentra Incorporation (INCORP)detennined at 68 hr. Radioactive precursor incorporation tions which produced mitogenesis and which reached maxi and enzyme assays were performed as described In â€˜MaterialsandMethods.â€• Actual PHA-stlmulated values were as follows. [3HJUrldlneIncorporation: 24 hr, mum levels at the time of maximum [3H]thymidine incorporation. 11.956 Â±1.094 (S.E.) cpm; 44 hr, 33,382 Â±1.440 cpm; 68 hr. 23,340 Â± Moreover, addition of the phosphodiesterase inhibitor, IMBX, 3,950 cpm. [â€˜Hjlhymidine Incorporation: 24 hr. 442 Â±271 cpm; 44 hr. 17,106 completely inhibited PHA-induced mitogenesis. Although the Â±3,245 cpm; 68 hr. 50,913 Â±13,412 cpm. Cyclic AMP phosphodlesterase specific activity: 0.670 pmol/mln/@g DNA. Cyclic GMP phosphodlesterase activ addition of this inhibitor resulted in an even greater elevation of Ity: 0.1 18 pmol/min/pg DNA. The stimulation ratios of PHA to control values cyclic AMP phosphodiesterase activity in the cells, one possi wereas follows.[3HJUrldlneincorporation:24hr. 6.4; 44 hr. 8.6; 68 hr. 17.7. ble explanation is that in vivo the enzyme is nonfunctional as [3HjThymldine: 24 hr. 1.1; 44 hr. 43.4; 68 hr, 98.4. CyclIc AMP phosphodlester ase: 5.2. CyclIcGMPphosphodiesterese:7.0.Resultsplottedrepresentthe long as IMBX is present, and the expression of more enzyme averageofduplicatedeterminationsofphosphodlesteraseactivityandtriplicate activity may be a compensatory response of the cell to this determinations of radioactive precursor incorporation. Although not shown, Iden enzyme inhibition, as has been observed for cultured fibroblast tlcal results were obtained when ptiosphodlesterase specific activity was deter mined as pmol/mln/g@gprotein. (30) and lymphoma cells (3). In serum-deprived, quiescent baby hamster kidney fibro blasts, we have found that serum and insulin produce a 10-fold of DNA synthesis (24, 25). The activation of phosphodiesterase activation of cyclic nucleotide phosphodiesterase, resulting in appears to be necessary for the cells to enter the S phase of a 3-fold lowering of cellular cyclic AMP content and stimulation the cell cycle, since inhibition of phosphodiesterase by IMBX

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phosphodlesterases. Adv. Cyclic Nucleotide Res., 3: 65â€”98,1973. inhibits this process (24). Lymphocyte activation requires in 2. Archer, S. B., Strade, S. J., Sanders,R. B., Pledger,w. J., Thompson,W. teraction with lectin during at least3 different stagesin order J., and Robison, G. A. Sonication as a tool for the study of adenylyl cyclase for the cells to progress through the steps necessary for activity. J. Cyclic Nucleotide Res., 3: 189â€”198,1977. 3. Bourne, H. R., Tomkins, G. M., end Dion, S. Regulation of phosphodlesterase entrance into the S phase of the cell cycle and for commitment synthesis: requirement for cyclic adenosine monophosphate-dependent pro to mitogenesis (45). Therefore, it is possible that in lympho tein kinase. Science (Wash. D.C.), 181: 952-954, 1973. cytes increased phosphodiesterase activity is required during 4. Byus, C. V., Klimpel, G. R., Lucas, D. 0., and Russell, D. H. Ornithine decarboxylase Induction in mitogen-stimulated lymphocytes is related to the lectin activationfor cellsto traversethe stepsrelatedto move specific activation of type I edenosine cyclic 3',5'-monophosphate-depend ment through G1 and entrance into S. This correlates concep ent protein kinase. Mol. Pharmacol., 14: 431 â€”441,1978. tually with observations that cyclic AMP and agents that raise 5. Cleland, W. W. The statistical analysis of enzyme kinetic data. Adv. Enzymol. Relat. Areas Mol. Blol., 29: 1â€”32,1967. intracellular levels of cyclic AMP inhibit lectin-induced mitogen 6. Coftey, R. G., Hedden, E. M., and Hadden, J. W. Evidence for cyclic GMP esis (7, 16, 33, 42). However, we have found that, despite the and calcium mediation of lymphocyte activation by mitogens. J. Immunol., 119:1387-1394,1977. activation of cyclic nucleotide phosphodiesterase activity by 7. DeRubertis, F. R., Zenser, T. v., Adler, W. H., and Hudson, T. Role of cyclic mitogens, there is no difference in basal cyclic AMP levels in edenosine 3',5'-monophosphate in lymphocyte mitogenesis. J. Immunol., control and in PHA-stimulated lymphocytes at 0, 1, 24, 48, and 113: 151â€”161,1974. 8. Desai, K., Marlan, M., and Appleman, M. M. Concanavalin A stimulation of 72 hr in culture. Others who have investigated cyclic AMP low K,@cyclic AMP phosphodiesterase of rat adlpocytes. Fed. Proc., 35: levels in human lymphocytes in response to PHA have found 1438, 1976. that following a very eariy, transient increase in cyclic AMP, 9. Douglas, S. D. Electron microscopicand functional aspects of human lymphocyte response to mitogens. Transplant. Rev., 11: 39â€”59,1972. the levels decline to the original basal level, or slightly lower, 10. Epstein, P. M., Mills, J. S., Ross, C. P., Strada, S. J., Hersh, E. M., and by 24 hr, and remainat that levelthrough72 hr in culture(23, Thompson, W. J. Increased cyclic nucleotide phosphodlesterase activity 32). Recently, however, in mouse splenic lymphocytes, it has associated with proliferation and cancer In human and murine lymphoid cells. Cancer Res., 37: 401 6â€”4023,1977. been shown that stimulation by Con A produces a 6-fold 11. Epstein, P. M., and Silverman, P. M. Induction of cyclic AMP phosphodies increase in cyclic AMP and a subsequent decline to its original terase in Blastocladiella emersonll and Its relation to cyclic AMP metabolism. J. Bacteriol., 135: 968-975, 1978. level just prior to the onset of DNA synthesis (40). Blockage of 12. Gillette, M. v., and Filosa, M. F. Effect of concenevalin A on cellular slime either the increase or the decline in cyclic AMP prevented mold development: premature appearance of membrane-bound cyclic AMP mitogenesis. phosphodiesterese. Biochem. Biophys. Res. Commun., 53: 1159-1166, 1973. A 10-fold induction of phosphodiesteraseactivity without 13. Greaves, M. F., and Bauminger, S. Activation of T & B lymphocytes by any change in basal cyclic AMP content has also been ob insoluble phytomitogens. Nat. New Blol., 235: 67-69, 1972. served during commitment to sporulation in the lower eukar 14. Hadden, J. W., Hedden, E. M., Sodlik, J. R., end Coffey, R. G. Effects of concanavalin A and a succinyleted derivative on lymphocyte proliferation yote, Blastocladiella emersonii (11). One possible explanation and cyclic nucleotide levels. Proc. Net. Aced. Sd. U. S. A., 73: 1717â€”1721, for this observation is that cyclic AMP may exist primarily in a 1976. 15. Hadden, J. W., Hedden, E. M., Heddox, M. K., and Goldberg, N. 0. bound state and thus be resistant to enzymatic hydrolysis (31). Guenosine 3',S'-cyclic monophosphate: a possible intracellular mediator of In this case, phosphodiesterase may be regulating a small, mitogenic influences in lymphocytes. Proc. NatI. Aced. Sd. U. S. A., 69: unbound pool and function primarily to return cyclic AMP levels 3024â€”3027,1972. 16. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1980 American Association for Cancer Research. Activation of Cyclic Nucleotide Phosphodiesterase from Isolated Human Peripheral Blood Lymphocytes by Mitogenic Agents

Paul M. Epstein, John S. Mills, Evan M. Hersh, et al.

Cancer Res 1980;40:379-386.

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