Induction of Interleukin 2 Production but Not Methionine Adenosyltransferase Activity Or S-Adenosylmethionine Turnover in Jurkat T-Cells1

[CANCER RESEARCH 52, 3361-3366, June 15, 1992] Induction of Interleukin 2 Production but not Methionine Adenosyltransferase Activity or S-Adenosylmethionine Turnover in Jurkat T-Cells1

James De La Rosa, Arthur M. Geller, H. Leighton LeGros, Jr., and Malak Kotb2

Veterans Administration Medical Center, Research Service. Memphis, Tennessee 38104 [J. D. L. R., H L. L., M. K.]; and the Departments of Surgery, Microbiology, and Immunology Â¡J.D. L. R., M. K.] and Biochemistry [A. M. G.J, University of Tennessee, Memphis, Tennessee 38163

ABSTRACT the reports that DNA methylation is important in the regulation of different lymphocyte genes upon cell activation and differ We have recently reported that methionine adenosyltransferase entiation (4-6). (MAT) in resting human peripheral blood I -colls is primarily present in Considering the importance of AdoMet in lymphocytes, it is the form of a precursor which we named X.This protein decreases upon cell stimulation, as both MAT activity and the amount of the catalytic Â«/ surprising that there is very little information regarding its rate a' subunits of the enzyme increase. When resting cells are activated by of utilization during the course of cell activation. One exception phytohemagglutinin, the decrease in X and increase in a/a' occurs after is the report by German et al. (7) which documented that after interleukin 2 (IL-2) production and before DNA synthesis. The human stimulation of PBMC with PHA, the AdoMet pool size and T-leukemia cell line, Jurkat, is unique in its ability to produce IL-2 in rate of AdoMet utilization increased (7). Subsequent studies by response to exogenous stimuli such as T-cell mitogens and therefore Kotb et al. (8) and De La Rosa et al. (9, 10) showed that upon provides a convenient model for studying biochemical reactions involved activation of PBMC, MAT activity also increased. More re in T-cell activation. In this study the regulation of MAT activity and .V- cently, we discovered that in resting human T-cells, MAT is adenosylmethionine (AdoMet) in resting and activated Jurkat cells was present in a precursor form called X (10). Upon lymphocyte investigated. Here we report that MAT activity in unstimulated Jurkat activation X, which has low MAT activity, disappears and the cells is about 10- and 3-fold higher than the activity in resting and a/a' subunits, which have high MAT activity, increase concom- activated peripheral blood mononuclear cells, respectively. Activation of itantly. The replacement of Aby a/a' subunits occurs after IL- Jurkat cells with phytohemagglutinin resulted in increased IL-2 produc tion, but not an increase in MAT activity. Identical results were obtained 2 synthesis and before DNA synthesis (10). using freshly isolated cells from acute lymphoblastic leukemia patients. The human leukemic T-cell line, Jurkat, provided cell biolo AdoMet utilization and pool size were approximately 3- and 10-fold gists with an excellent tool to study events of T-cell activation higher, respectively, in Jurkat cells compared to peripheral blood mono- in a homogeneous population of cells because of its unique nuclear cells, and both parameters were unaffected by phytohemaggluti ability to produce IL-2 in response to mitogens and certain nin stimulation. Jurkat MAT was determined to be structurally indistin superantigens (11). We have used this T-cell line to study the guishable from enzyme from T- or B-leukemia cells but was different regulation of MAT activity and AdoMet metabolism in lym from resting, normal I-rolls in that it lacked the X form. Furthermore, unlike MAT in resting T-cells, the relative amounts of the a, a', and ÃŸ phocytes. We report here that in Jurkat cells, unlike resting normal human T-cells, activation is not accompanied by either subunits of the enzyme did not change throughout the course of IL-2 induction. We conclude that AdoMet metabolism and MAT activity in a change in the relative amounts of M AT subunits or an increase Jurkat cells are constitutive!}' high and that induction of IL-2 synthesis in MAT activity, AdoMet levels, or AdoMet turnover. AdoMet in these cells is independent of changes in AdoMet synthesis or turnover. levels and MAT activity are constitutively higher in Jurkat cells The lack of the X form and the difference in MAT regulation between compared to either resting or activated T-cells. The differential leukemic T-cells and peripheral blood mononuclear cells may be exploited regulation of MAT in normal and leukemic T-cells may in part in the design of specific chemotherapeutic agents. be due to the fact that Jurkat cells lack the X form of the enzyme. INTRODUCTION MATERIALS AND METHODS AdoMet3 donates methyl groups to proteins, nucleic acids, and various lipids and donates propylamine groups for the Materials. Aprotinin, antipain, chymotrypsin, leupeptin, pepstatin A, soy bean trypsin inhibitor, benzamidine. o-phenanthroline, phenyl- biosynthesis of polyamines. The importance of AdoMet in methylsulfonyl fluoride, PHA, bicinchoninic acid, Ficoll-Hypaque, and proliferating cells is underscored by the well established role the iodide salt of AdoMet were from Sigma Chemical Co. (St. Louis, polyamines play in proliferation, the recently discovered role of MO). The tissue culture media and fetal bovine serum were from Gibco protein methylation in the processing of ras proteins (1,2), and BRL (Gaithersburg, MD). L-["S]Methionine was purchased from Du Pont New England Nuclear (Wilmington, DE), and the chemilumines- Received 11/20/91; accepted 4/7/92. cence detection system (ECL) was from Amersham (Arlington Heights, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in IL). Nitrocellulose membranes and horseradish peroxidase-conjugated accordance with 18 U.S.C. Section 1734 solely to indicate this fact. goat anti-rabbit polyclonal antibody were from Bio-Rad (Richmond, 1This work was supported in part by grants from the United States Veterans CA) and XAR-5 film was from Kodak (Rochester, NY). Administration (M. K.), and from the NIH, GM-38530 (M. K.). The ALL-2 cells were obtained from The Leukemia Cell Bank (LCB) at St. Jude's Children's Leukemic T-Cells. Freshly isolated ALL-2 cells (T-cells, midthymo- Research Hospital with the assistance of Dr. Fredrick Behm. The LCB is cyte) from pediatrie patients were provided by Dr. F. Behm at St. Jude supported by the National Cancer Institute and by funds from the American Children's Research Hospital. The Jurkat cells were from the American Lebanese and Syrian Associated Charities. 2To whom requests for reprints should be addressed, at the V. A. Medical Type Tissue Culture Collection (Rockville, MD). Cells were cultured in RPM1 supplemented with 10% fetal bovine serum, 2 mM L-gluta- Center (151), 1030 Jefferson Ave., Memphis, TN 38104. 3The abbreviations used are: AdoMet, S-adenosy Imethionine: PBMC, periph mine, 50 ng/ml of streptomycin, and 50 units/ml of penicillin (referred eral blood mononuclear cells; MAT, methionine adenosyltransferase (ATP:L- to as RPMI complete). All cells were cultured at 37Â°Cin humidified methionineS-adenosyltransferase. EC 2.5.1.6); PHA, phytohemagglutinin; SDS- air containing 5% CO?. PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PBS, phos phate buffered saline ( 140 mM NaCl, 2.7 mivi KCI, 1.5 mM KH2PO4, and 8.1 mivi Preparation of Cell Extracts. Cell extracts were prepared for analysis NaÂ¡HPCX,pH 7.4); HPLC, high performance liquid chromatography; LDH, of AdoMet and L-methionine by lysing cells, precipitating protein with lÃ¡clatedehydrogenase; IL-2, interleukin 2; PMA, phorbol myristic acetate. 2 N perchloric acid, and then neutralizing the extract with KOH and 3361

KHCOj. as described previously (7-9). and the specific radioactivity of the AdoMet was determined by HPLC MAT activity was assayed from extracts prepared by three cycles of and liquid scintillation counting. freeze-thawing. The enzyme extraction buffer contained 50 mM potas Other Methods, Activity Units, and Kinetics. MAT and LDH were sium yV-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, pH assayed by the methods of Kotb and Kredich (12) and Bergmeyer and 7.4; 50 mM KCI; 15 mM MgCl2; 0.3 mM EDTA; and the following Bern t (13), respectively. Protein was determined spectrophotometri- proteolytic inhibitors: 0.5 mM phenylmethylsulfonyl fluoride, 1 mM o- cally by the bicinchoninic acid method (14), and IL-2 was determined phenanthroline; 10 mM benzamidine; 300 Mg/ml soy bean trypsin by the CTLL bioassay ( 15). Ten units of IL-2 is defined as that amount inhibitor; 50 Mg/ml aprotinin; 1 Mg/ml pepstatin A; 25 Mg/ml antipain; which will stimulate a proliferative response of the CTLL cell line to 25 Mg/ml chymostatin; and 25 Mg/ml leupeptin. If the extract was to be 50% of the maximum obtained with a standard preparation of IL-2. analyzed by SDS-PAGE, the proteolytic inhibitors were dissolved in Proliferation was determined by the incorporation of pHjthymidine 50 mM Tris, pH 7.4-50 mM NaCl-5 mM MgCl2. into precipitable radioactivity. L-Methionine and AdoMet Turnover Studies. The isotope kinetic One unit (U) of MAT activity is that amount of enzyme which will model described by German et al. (7) was used to determine the catalyze the formation of 1 nmol of AdoMet in 1 h, whereas 1 unit of fractional turnover of intracellular L-methionine (k\) and AdoMet (k2). LDH is that amount of enzyme which will catalyze the formation of 1 The model showed that if the specific radioactivity of extracellular Mmol of lactate in 1 min. methionine, denoted by MI,is known, then the time-dependent change SDS-PAGE and Western Blotting. Samples were dissolved in loading in the specific radioactivity of intracellular methionine, denoted by M2. buffer (60 mM Tris-Cl, pH 6.8; 2% SDS; 5% 2-mercaptoethanol; and in a culture of cells can be described by Equation A. Similarly, the time- 5% glycerol) and heated in a boiling water bath for 3-4 min. The SDS- dependent change in the specific radioactivity of AdoMet, denoted by PAGE resolving gel consisted of 10% acrylamide and 0.27% bisacry- MS,can be described by Equation B. lamide. Electrophoresis was done at 15-20 V/cm with cooling. The characterization of antibodies produced to human lymphocyte MAT (A) has been published recently (16). Secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit polyclonal antibody. The trans I. *2g-*" fer for Western blotting was done for 1 h at 25 to 30 V/cm with cooling. MJ = Ml M - . _ . (B) k2-k. The blots were blocked overnight with 6% nonfat milk in 50 mM Tris, pH 7.5-150 mM NaCI. After incubating the blot with primary and In these experiments MI,m, and MJwere determined empirically over secondary antibody, immunoreactive proteins were detected with the time so that the turnover constants of kÂ¡and A2could then be deter luminol-chemiluminescence system (ECL) from Amersham. The proc mined graphically by iteration. In most experiments, the specific radio essed blot was sandwiched between two overhead transparencies and activity of intracellular L-methionine (MZ)and AdoMet (MJ)approached exposed to XA-R film for 10 to 60 s. The limit of detection under these asymptotes which ranged from 80 to 99% of the value for extracellular conditions was about 2 ng for each of the human lymphocyte MAT methionine (MI).A similar observation in PBMC was made previously subunits. (7) and suggests the presence of metabolically inaccessible pools of AdoMet and methionine. It was these asymptote values which were used in modeling to obtain A, and Ac2. RESULTS The specific activity of intracellular and extracellular L-methionine was determined after adding 2.5 to 3.0 MCiof L-["S]methionine to snap The Jurkat human T-cell line was studied for two reasons: top vials containing a culture of IO7cells in 1 ml of RPMI complete. (a) T-cells respond to PHA and are responsible for the changes The vials were kept in a 37Â°Cwater bath and at specific times, within observed in AdoMet metabolism and MAT activity in PHA- 90 s, 0.8 ml of culture was removed and diluted into 50 ml of ice cold activated PBMC; (b) although Jurkat is a transformed cell line PBS. Both the vial and the conical centrifuge tube containing the which proliferates continuously, it is considered a relatively diluted cells were then centrifuged for 1 min at 1800 x g. The medium resting T-cell line because, unlike other T-cell lines, it can be in the vial was collected and used to determine the specific radioactivity induced to secrete IL-2. Therefore, it was reasoned that because of extracellular L-methionine. The cells in the conical tube were washed Jurkat cells can be activated, they might serve as a model to briefly with ice cold PBS, frozen in liquid nitrogen, and stored at -70Â°C study the role of AdoMet metabolism in the induction of IL-2 until extracted (as described above) for intracellular methionine. En synthesis. dogenous AdoMet was removed from the cell extract by adsorption to Induction of IL-2 Synthesis in Jurkat Cells Is Not Accom acid washed charcoal, which was added to the extract at 65 Mg/106 cells. Both extracellular and intracellular L-methionine in the extracts panied by an Increase in MAT Activity. Jurkat cells were incu bated with different concentrations of PHA to determine the were converted enzymatically to AdoMet. To convert methionine to optimal concentration for cell activation, as indicated by IL-2 AdoMet, extract was incubated with 100 mM Tris (pH 8.3), 100 mM KCI, 20 mM MgCl2, 2.5 mM ATP, 2 mM dithiothreitol, 150 Mg/ml of production, and for the induction of MAT activity. This dose bovine serum albumin, and 10 Mg/ml of Escherichia coli MAT for l h response experiment demonstrated that IL-2 production by the at 37Â°C.Following this conversion, a cell extract was made as described Jurkat cells increased concomitantly with increasing PHA con above. AdoMet was then separated from other radiolabeled compounds centration, reaching a maximum at 2.5-5 Mg/ml (Fig. 1/4). by HPLC using a Whatman SCX strong cation exchange column (0.46 However, MAT activity did not increase upon Jurkat activation, x 25 cm) described by German et al. (7), with the modification that the as was previously found for PBMC, and in fact appeared to mobile phase was 0.2 M ammonium formate, pH 4, containing 10% decrease (Fig. IB). LDH, which is a noninducible enzyme, was acetonitrile. (-)AdoMet was purified chromatographically (12) and used as a negative control, and its activity remained relatively used as the HPLC standard. The radioactivity in the AdoMet-peak constant upon activation (Fig. 1C). from each HPLC run was measured by liquid scintillation counting. Freshly Isolated Leukemic T-Cells Behave Like Jurkat Cells. The specific radioactivity of AdoMet was then calculated as the ratio Next we asked whether the lack of induction of MAT activity of cpm to nmol of AdoMet. represented an idiosyncratic behavior of the Jurkat clone or a The equilibration of radioactivity into AdoMet produced by the cells was determined similarly. Jurkat cells, at a cell density of 2 x IO6cells/ general characteristic of leukemic T-cells. Freshly isolated PBMC from patients with ALL-2 were incubated for various ml, were incubated with radiolabeled methionine at 3 nC\/m\ for up to 2 h. At specific times 3 x IO6 cells were harvested, centrifuged for 1 times with optimal mitogenic concentrations of PMA plus min at 1800 x g, washed with PBS, and frozen as a cell pellet in liquid ionomycin and then tested for IL-2 production and MAT nitrogen until extracted. Cell extracts were made as described above, activity. PMA and ionomycin were used instead of PHA be- 3362

MAT Subunit Composition in Resting and Activated Jurkat Cells. Recent data from our laboratory revealed the presence of a precursor of MAT in resting T-cells. This M, 68,000 precursor (X) which has low MAT activity disappears when T-cells are stimulated with PHA while the Â«and Â«'subunits of the enzyme which have high MAT activity increase. The amount of the ÃŸ subunit, on the other hand, remained constant throughout the 20 course of stimulation. These changes in MAT subunits always occurred after IL-2 production and before DNA synthesis. It 0.0 1.0 2.0 3.0 4.0 5.0 was of interest, therefore, to determine whether the induction PHA Concentration, ng/ml of IL-2 production by Jurkat cells is also accompanied by a change in the relative amounts of MAT subunits. Cell extracts were prepared from resting and 24-h PHA-stimulated Jurkat 50 e cells and analyzed in Western blots for MAT subunits. In S 40 contrast to normal T-cells, Jurkat cells as well as ALL-2 cells lacked the Xform and only had the a, a', and ÃŸsubunits (data I 30 not shown). Furthermore, when the cell extracts from the 24-h time point of the dose response experiment in Fig. 1 were analyzed by Western blotting, there was no change in the relative amounts of the different MAT Â«,a', and ÃŸsubunits (Fig. 2). There was a slight but insignificant increase in the ÃŸ 0.0 I 0 2.0 3.0 4.0 5.0 subunit. PI IA Concentration, ng/ml AdoMet Utilization in Resting and Activated Jurkat Cells. Although the above experiments showed that MAT activity did not increase upon activation of Jurkat cells, this did not pre clude the possibility that AdoMet utilization or pool size could change. To estimate the absolute rate of AdoMet utilization in stimulated and unstimulated Jurkat cells, the equilibration of extracellular radiolabeled methionine with intracellular methionine and AdoMet was determined. The time course of this equilibration is shown in Fig. 3. In both stimulated and unstim < ulated Jurkat cells methionine specific radioactivity reached half-maximum in about 15 s (Fig. 3/1). The equilibration of radiolabel with AdoMet reached half-maximum within 24 min. 1.0 2.0 3.0 4.0 5.0 Both the specific radioactivity of methionine and AdoMet ap PHA Concentration, (Jg/ml proached an asymptote between 80 and 99% of the specific Fig. 1. PHA dose response of IL-2 production (A). MAT activity (B). and LDH activity (C). Jurkat cells at a density of 4.0 x 10' cells/ml were incubated activity of extracellular methionine. with different concentrations of PHA and the cells were harvested after either 10 The fractional turnover of intracellular methionine (k,) and h (O) or 25 h (â€¢).The media were assayed for IL-2 and the cell extracts were AdoMet (&:) was determined graphically from these equilibra assayed for both MAT and LDH activity. tion curves using Equations A and B under "Materials and Methods," and these values are listed in Table 2. Included in

Table 1 Induction of IL-2 synthesis but not MAT activity in freshly isolated ALL- Table 2 are the values from stimulated and unstimulated PBMC 2 cells reported previously (7). The fractional turnover of L-methionine ALL-2 cells were cultured for 24 h at 2 x 106/rnl Â¡nthe presence or absence was similar in both stimulated and unstimulated Jurkat cells of 50 ng/ml phorbol myristate acetate and 0.5 ^M ionomycin. These concentra ranging from 2.5 to 4.0 min"1, which is similar to that for tions were determined in a separate experiment to be optimal for IL-2 production. PBMC (2.5 min"1; Table 2). Upon cell activation, the AdoMet After 24 h the cells Â»ereseparated from the culture media by centrifugation. The culture media were assayed for IL-2 activity, and the cell pellet was extracted and assayed for MAT activity and protein concentration as described in "Materials and Methods."

activity Incubation time activity (units/mg a1a, (h)Zero (units/ml)3.6 protein)11.3 time 24(-) 10.3 24(+)IL-2 118MAT 12.6 -ÃŸ cause a number of these cells showed aberrant CD3 expression 0 0.25 1.0 2.5 5.0 std which can interfere with PHA responsiveness. As shown in Table 1, when freshly isolated T-leukemic cells were stimulated PHA concentration, jig/ml with PMA and ionomycin, the IL-2 production increased from Fig. 2. Western blot analysis of Jurkat cell extracts stimulated with different less than 4 units/ml to greater than 100 units/ml after a 24-h concentrations of PHA. The first five lanes correspond to the 25-h samples from Fig. 1. and the last lane is a MAT standard (std) purified from chronic lymphocytic incubation. However, as the case with Jurkat cells, MAT activ leukemia cells. The blot was probed with anti-holoenzyme antisera, and processed ity remained essentially the same. as described in "Materials and Methods." 3363

fractional turnover increased in PBMC by 38-200%; however, 1.0 in Jurkat cells, AdoMet turnover was not significantly affected 0.8 (Table 2). After activation of PBMC, the pool size of AdoMet also increased 4-10 fold compared to the unactivated cells 0.6 (Table 2). On the other hand, the pool size of AdoMet in Jurkat 0.4 cells remained constant at 140 pmol/106 cells, regardless of whether the cells were activated or not (Table 2). Although the 0.2 fractional turnover of AdoMet in Jurkat cells was slightly less than that of PBMC, the fact thai the AdoMet pool size in 0.00 0.25 0.50 0.75 1.00 1.25 1.50 Time, min Jurkat cells (184 ^M) was at least 23 times that of unstimulated PBMC (3-8 MM)indicated that AdoMet utilization in Jurkat cells was higher than in PBMC. In fact, AdoMet utilization in Jurkat cells was 5-7 ^M/min compared to a value of 0.12-0.4 1.0 and 1.5 /Â¿M/minin unstimulated and stimulated PBMC, re 0.8 spectively (Table 2). Correlation between Predicted and in Situ MAT Activity in 0.6 Resting PBMC versus Stimulated PBMC and Jurkat Cells. Using the steady state kinetic model of Kotb and Kredich (17) 0.4 describing the MAT catalyzed reaction, we predicted the MAT 0.2 activity in both PBMC and in Jurkat cells (Table 3). Enzyme activity can be predicted as a fraction of the Vmaxif the concen 0.0 50 75 100 trations of the substrates and products of the reaction are Time, min known. The Vmaxitself was estimated empirically by assaying Fig. 3. Time course of equilibration of extracellular i.-["S]mcthionine with intracellular i.-methionine and AdoMet in unstimulated and stimulated Jurkat T- Table 3 Predicted MAT reaction velocity and predicted MAT activity in PBMC cells. After the addition of radiolabeled methionine, cells were harvested at the and Jurkat T-cells indicated times and the specific radioactivities of extracellular methionine (nÂ¡), The MAT activity of unstimulated (-PHA) and stimulated (+PHA) PBMC intracellular methionine (nÂ¡).and AdoMet (n,) was determined as described in "Materials and Methods." Equilibration as defined here is the MI^I ratio for i.- and Jurkat cells was predicted using a previously published steady state kinetic methionine and M.VMIforAdoMet. A, equilibration of radiolabel into I.-methionine model describing the enzyme activity (16). in unstimulated (D) and stimulated cells (â€¢).B, equilibration of radiolabel into PBMC Jurkat cells AdoMet in unstimulated (O) and stimulated cells (â€¢).Theopen and closed symbols -PHA represent actual data points, while the curves represent theoretical cunes modeled +PHA -PHA +PHA to fit the data using Equations A and B in "Materials and Methods." In I the Predicted reaction velocity 0.49 0.28 0.09 0.09 dashed line is to data from unstimulated cells modeled to 85% full equilibration expressed as a fraction of resulting in A, = 3.0 min "', and the solid line is to data from stimulated cells Vmâ€žÂ° modeled to 99% full equilibration resulting in A, = 2.5 min ~*. In B the dashed Predicted MAT activity* 0.36-0.49 0.56-0.76 2.1-2.7 1.7-2.4 line for data from unstimulated cells using k, = 3 min "', modeled to 95% full (pmol/min/10'cells) equilibration resulting in A2= 0.035 min "'; and the solid line is to data from In situ MAT activity' 0.05-0.17 0.97-1.08 3.5-5.0 3.1-4.2 stimulated cells using A. = 2. modeled to 95% full equilibration and resulting in (pmol/min/10'cells) Aj = 0.025 min ~'. a The predicted reaction velocity for PBMC was estimated using the steady state kinetic model of Kotb and Kredich (16) shown below, where K, is Vmâ€ž,vis the initial velocity. A is the ATP concentration. B is the i -methionine concentra tion, P is the AdoMet concentration, Q is the pyrophosphate concentration, and R is the phosphate concentration. Table 2 Comparison of AdoMel turnover, pool si:e, utilisation, and MAT activity in PBMC y,AB(\ + dQ + gQR) Unstimulated (-PHA) and stimulated (+PHA) PBMC and Jurkat cells were A'â€žÃ„+AB + Câ€žP+ Câ€žAP(â€”T-H incubated with radiolabeled methionine to determine the fractional turnover of \ A,Â»/ u-methionine (A,) and AdoMet (Aj). The AdoMet content of the cells was measured by HPLC. and MAT activity was determined by a radiochemical assay. â€ž¿/l+ W One unit of MAT activity catalyzes the formation of 1 nmol of AdoMet/h. A',0AÂ» PBMCA, + câ€ž (min"') A2(min~')AdoMet 0.029-0.06Â°1.4-3.4 I + Kw\ 0.04-0.09117-230.025-0.0361400.022-0.03140 C,rQR d + Câ€ž,PQR pool size pmol/ 10' cells jimol/min*AdoMet 3.3-8.10.05-0.1725-330.97-1.08 1843.5-5.0 1843.1-4.2 The values obtained from the report of De La Rosa et al. (9) of 5 UMpyrophos phate. 5 HIMphosphate. 1.5 HIMATP, 20 /*Mmethionine, and 5 or 30 nM AdoMet utilization pmol/min/10' cells Â»ereused in solving for v. The same values were used for predicting i- Jurkat cells, except that methionine was 100 >iM(concentration in RPMI), and AdoMet Â¿imol/minMAT 0.12-0.42-5f+PHA2.51.4-1.58-15Jurkal-PHA2.7-4.04.6-6.630-40cells+PHA2.5-3.54.1-5.525-35 was calculated to be 184 ^M, based on a measured cell volume of 0.76 ml/10' cells. activity (units/ * This value is the product of the predicted reaction velocity (fraction of Vâ€žâ€ž) mg protein)-PHA2.5 and the enzyme activity assayed under conditions of near-substrate saturation, Â°Turnover estimates for PBMC arc from report of German et al. (7). after it was corrected to 100% activity under total substrate saturation. Unstim * AdoMet pool size (nmol/ml cell volume) and AdoMet utilization (nmol/ ulated and 24-h-stimulated PBMC have activities of 0.73-1.0. and 2.0-2.7 pmol/ min/ml cell volume) were calculated based on cell volume for stimulated and min/10' cells, respectively. Unstimulated and stimulated Jurkat cells have activ unstimulated PBMC of 0.42 ml/10' cells and 0.69 ml/10' cells estimate by ities of 23-30 and 19-26 pmol/min/10* cells, respectively. German et al. (7). The cell volume for (he clone of Jurkat cells used in these ' The in situ MAT activity is the rate of AdoMet utilization (listed in Table 2), studies was calculated to be 0.76 ml/106 cells. since under steady state conditions the rate of AdoMet utilization should equal ' MAT activities for PBMC are from De La Rosa et ai (9). its rate of svnthesis. 3364

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1992 American Association for Cancer Research. MAT ACTIVITY AND AdoMel METABOLISM IN JURKAT CELLS the enzyme under conditions of near-substrate saturation. Thus, a/a' form (10). Although this process is detected by 24 h of the predicted MAT activity in situ is simply a product of the cell activation, it reaches a maximum at 48 h following IL-2 fraction of Vnux and the assayed MAT activity (which was production and preceding DNA synthesis. It was not clear from corrected for 100% substrate saturation). Under steady state our study whether the change in these subunits is a result or conditions it is expected that the rate of AdoMet utilization prerequisite of the cell cycle GÃ¬to S transition. As shown in would equal the rate of input, i.e., the in situ MAT activity. In this study Jurkat and ALL-2 cells lack the X form, and the unstimulated PBMC, the in situ MAT activity was 7-3 fold enzyme is constitutively present in the highly catalytic form a/ Â«'.This may explain the lack of induction of enzyme activity lower that the predicted value. In contrast, in situ MAT activity in stimulated PBMC, as well as in Jurkat cells, was 2-fold despite induction of high levels of IL-2. Again the issue of higher than the predicted MAT activity (Table 3). whether this constitutively activated AdoMet metabolism is a cause or result of the process of malignant transformation requires further experimentation. DISCUSSION Clearly intact AdoMet metabolism is essential for normal T- Abnormalities in methionine metabolism have been reported cell proliferation. Previously, we demonstrated that inhibitors in a variety of cancer cells (18-21). Whether these changes are of AdoMet synthesis block T-cell blastogenesis in response to a consequence or cause of malignant transformation is not mitogenic and superantigenic stimulation (8). This is consistent clear. However, inhibitors of AdoMet synthesis or inhibitors of with clinical and experimental reports that lymphocytic cells DNA methylation have been shown to induce the differentiation depend on AdoMet mediated reactions for immune function (32-35), which is not surprising since these cells undergo rapid of several transformed cell lines (22, 23). As a result of these reports, several laboratories have dedicated their efforts to differentiation. Indeed, the role of DNA methylation and the studying the metabolism of AdoMet and designing specific regulation of the expression of certain genes in lymphocytes has been shown in several studies (4-6). A comparison between inhibitors of its metabolism for use as chemotherapeutic agents (24-28). We are particularly interested in the role of AdoMet the predicted and in situ MAT activity in resting and stimulated in the activation and differentiation of human T-lymphocytes T-cells showed some interesting correlations. In unstimulated and therefore have engaged in studies of the regulation of MAT cells the in situ MAT activity was lower than the predicted activity in normal resting, activated, and transformed T-cells. activity, but in stimulated T-cells and Jurkat cells the situation Although several studies have compared MAT activity and was reversed. These observations can be interpreted in several ways: (a) Resting T-cells may have a less active form of MAT AdoMet turnover in normal and malignant cell lines (21, 29, compared to stimulated cells; (b) an inhibitor of MAT may be 30) the data are rather confusing. In some cases there was no present in resting but not in stimulated T-cells; (c) activation difference in AdoMet metabolism, while in others, MAT spe of T-cells may be accompanied by induction of a MAT activa- cific activity was much higher in the transformed cells. We have found that both immortal T- and B-cell lines have much higher tor(s). None of these possibilities are mutually exclusive, and MAT specific activity than activated normal T- or B-lympho- further studies should reveal the mechanism of MAT regula tion. However, the finding in this study that the differences in cytes or freshly isolated leukemic cells. However, MAT specific activity varied depending on the lineage of the leukemic cells.4 AdoMet metabolism are at least in part related to the presence The Jurkat cell line has been used in many studies of T-cell of different forms of MAT in quiescent and actively dividing T-cells raises the possibility that this may be a feature that can activation because unlike other transformed T-cell lines, this be exploited for chemotherapy and immunomodulation. line is able to respond to mitogenic stimuli by producing high levels of IL-2. These Jurkat cells, as shown in this study had a Previous reports by Liau et al. (36) showed that tumor cells resting level of MAT activity of 30-40 units/mg protein and may have a different form of MAT. Such differences between AdoMet utilization rate was 20-55 times that of unstimulated normal and transformed cells provide the impetus for a more PBMC, and 3-5 times that of stimulated PBMC. Activation of rigorous search for selective inhibitors of the various forms of MAT that can be of potential benefit in chemotherapy. Of Jurkat cells by PHA had no effect on either MAT activity or particular interest in this regard is the report by Sufrin and AdoMet turnover. This was not unique to this cell line because freshly isolated ALL-2 cells when stimulated to produce IL-2 Lombardini (37) which showed, based on studies with several substrates and inhibitors of MAT, that there are differences in behaved the same way. These results are similar to those ob the active-site of tumor versus normal isozymes of MAT. It tained by Chiba et al. (31 ) with the promyelocytic cell line, HL- may be possible, therefore, to synthesize inhibitors specific for 60 cells. 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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1992 American Association for Cancer Research. Induction of Interleukin 2 Production but not Methionine Adenosyltransferase Activity or S-Adenosylmethionine Turnover in Jurkat T-Cells

James De La Rosa, Arthur M. Geller, H. Leighton LeGros, Jr., et al.

Cancer Res 1992;52:3361-3366.

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