242
Biochimica et Biophysica Acta, 1127 (1992) 242-248 © 1992 Elsevier Science Publishers B.V. All rights reserved 0005-2760/92/$05.00
BBALIP 53968
Phospholipase D activity in nontransformed and transformed fibroblasts Chunfa Huang ~ and Myles C. Cabot Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Medical Center Boulevard, Winston.Salem, NC (USA)
(Received 28 February 1992)
Key words: Phospholipase D; Fibroblast; Transformed cell
R~t embryo fibroblasts (REF$2cells) and the simian virus 40 transformed derivative (WT6 Ag6) were employed to characterize phospholipase D (PLD) activity in normal and t~ansformed cells, in cells prelabeled with [3H]myristic acid or [3H]glycerol and treated with I2-O-tctrad¢canoylphorbol-13-acetate (TPA, 50 ng/ml medium) or vasopressin (VP, 100 ng/ml medium) in the presence of ethanol, the formation of labeled phosphatkiylethanol (PEt) was 3- to 5-fold higher in REF52 cells than in the transformed cells. The transphosphatidylation of phosphatidylcholine (PC) to PEt was further examined in cell-free assay systems. Results demonstrated that the formation of PEt in the cell-free assays was dependent on the mode of substrate presentation and the source of the PC. With endogenous membrane-bound substrate, the formation of [aH]myristoyi-PEt was 5-fold higher in homogenates derived from normal cells as compared to transformed cell homogenates. In experiments using exogenous labeled PC isolated from either REF52 or transformed cells as substrate, cell-free PLD activity differed greatly with regard to the source of the PC. The formation of PEt from REF52-derived PC was approx. 4-fold higher as compared to PEt formed with PC derived from the transformed cells, irrespective of enzyme source. The results demonstrate that PLD in intact nontransfotmed fibroblasts is activatable by TPA and VP to a greater extent than in the transformed counterpart. The results from cell-free assays suggest that PLD activity is more dependent on the type of PC substrate than on the source of the enzyme.
Introduction DAG, formed via phosphoinositide hydrolysis by PLC, is an important second messenger that promotes translocation and activation of protein kinase C [1,2], DAG is also a pivotal intermediate in cellular lipid metabolism. New evidence has recently shown that DAG in agonist-stimulated cells, can likewise be derived from PC by receptor-dependent activation of PLC and PLD [3-5]. PLD (EC 3.1.4.4) catalyzes the breakdown of the terminal phosphodiester bond of
C~dence to: M.C. Cabot, Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Medical Center Boulevard, Winston-Salem, NC 27157-1016, USA. t Visiting scientist from Xiamen University, Department of Biology, Xiamen, People's Republic of China. Abbz~iatiom: In the naming of the glyceropbospholipids, we have ~ distinguished between the type of aliphetic linkage to glycerol (es~r or ether). D&G, diacylllycerol; PLC, phospholipase C; PLD, p~,osO~lipzse 13;,PEt, phesphatidylethanol; REF$2, rat embryo ft. broblasts; SV4O, simian virus 40; TIPA, 12-O-tetradecanoylphorbol13-acetate; VP, vasopressin; BSA, bovine serum albumin; FIBS, fetal bovine serum; TLC, thin-layer chromatography; PC, phosphatidylcholine; TAG, triacylglycerol; PBS, phosphate-buffered saline.
glycerophospholipids to produce phosphatidic acid and the relevant bases. Phosphatidic acid, like DAG, is not only a key intermediate in recycling of the glycerol backbone [6], but may as well function as an element of cellular signal transduction [7]. In addition, phospha. tidic acid can be hydrolyzed to DAG by phosphatidic acid phosphatase. By measuring the formation of phosphatidic acid or PEt, several research groups have demonstrated the presence of agonist-stimulated PLD activity in nontransformed cultured animal cells and tissues [8-12]. Few studies have been undertaken to assess PLD activity in transformed cells, although some :~tudies have demonstrated increased DAG levels, via a mechanism attributed to PLC action in ms-transformed cells [1316]. In the present study rat embryo fibroblasts (REF52) and a SV-40 transformed derivative (WT6 Ag6) [17,18] were used to examine the relationship of cellular transformation to PLD activity. Herein, we have characterized for the first time, PLD activity in fibroblasts of nontransformed and transformed origin. The results reveal that, although agonist-induced PLD activity was higher in the nontransformed fibroblasts, when assessed by cell-free assay, enzyme activity was shown to be strongly dependent on the source/type of PC sub-
243 strate. It is hoped that these results will set the stage for future research on the role of PC and the action of PLD in cellular proliferation. Materials and Methods
Materials. [9,10(n)-3H]Myristic acid (39.3 Ci/mmol), [2-3H]glycerol (10 Ci/mmol) and [methyl-3H]choline chloride (86.7 Ci/mmol) were purchased from New England Nuclear (Boston, MA). [9,10(n)-3H]Stearic acid (60 Ci/mmol) was from American Radiolabeled Chemicals (St. Louis, MO). TPA was supplied by Chemicals for Cancer Research (Eden Prairie, MN). Cell culture media were purchased from Gibco (Ghithersburg, MD). [ArgS]VP and bovine serum albumin (BSA) were from Sigma (St. Louis, MO). Fetal bovine serum (FBS) was obtained from HyCIone (Logan, UT). Silica gel TLC plates were from Analtech (Newark, DE), and solvents were purchased from Burdick and Jackson (Muskegon, MI) and J.T. Baker (Phillipsburg, N J). Lipid standards for TLC were purchased from Avanti Polar Lipids (Alabaster, AL) and Nu Chek Prep (Elysian, MN). Cell culture, radiolabeling and treatment. The rat embryo-derived fibroblasts (REF52) and the SV40 transformed tumorigenic variant (WT6 Ag6), provided by Dr. D.B. McClure (Division of Molecular and Cellular Biology, Eli Lilly and Company, Indianapolis, IN), were cultured as previously described [17,18]. REF52 ceils were used at passage 8-18, and WT6 Ag6 cells were used at passage 15-18. Cells were labeled with [3H]myristic acid (1 /zCi/ml of 5% FBS medium) or [3H]glycerol (5/zCi/ml of 5% FBS medium) for 24 h. After removal of labeling medium, cultures were rinsed twice in serum-free medium containing 1 mg BSA/ml and further equilibrated by incubation in the same medium for 1 h ([3H]myristic acid-labeled cells) or 2 h ([3H]glycerol-labeled cells). Cells were treated with either TPA (50 ng/ml) or VP (100 ng/ml), as indicated in figures, in medium containing 2% ethanol. Experiments were terminated by extracting total cellular lipids using the Bligh and Dyer procedure [19], modified to contain 2% acetic acid in methanol. Radiolabeled PC substrates, subceUular fractionation, and cell.free reaction conditions. To prepare radiolabeled PC, REF52 cells were labeled with either [3H]stearic acid (20/~Ci/ml of 5% FBS medium) or [3H]choline (15/~Ci/ml of 5% FBS medium) for 48 h. Transformed cells were prelabeled with [3H]stearic acid (20 ~Ci/ml of 5% FBS medium) for 48 h. Cultures were then rinsed twice in serum-free medium (1 mg BSA/ml) and equilibrated for 2 h in the same medium. Total cellular lipids were extracted [19] and PC was resolved by preparative TLC using a solvent system containing c h l o r o f o r m / m e t h a n o l / a c e t i c acid/water (50: 27: 8: 2.5, v/v). The PC fraction was
eluted from the silica gel by a modified Bligh and Dyer procedure [19], in which a 5 ml volume of methanol/10% acetic acid/water (25: 8: 4, v/v) was added. After vortex mixing, the sample was set aside for 10 min and then 5 ml chloroform, 1.6 ml methanol, and 3.5 ml water were added. Samples were mixed and centrifuged, and the lower phase was filtered through glass wool-plugged pipets and evaporated with nitrogen. The radiospecific activity of substrates in labeled membranes and in labeled exogenous PC ([3H]stearoyl-PC from REF52, 18.3 t~Ci//~mol; phosphatidyl[3H]choline from REF52, 23.4 t~Ci/t~mol; [3H]stearoyl-PC from WT6 Ag6, 20.9 t~Ci/t~mol) was determined by phospholipid phosphorus analysis [20]. Subct:llular fractions were prepared essentially according to the procedure of Martin [21]. Briefly, cultures were washed three times with cold Ca 2+. and Mg2+-free PBS (pH 7.2). Cells were scraped in ice-cold 10 mM Hepes/154 mM NaCl/1 mM EDTA (pH 7.5) and sonicated for three 10-s intervals. The cell sonicates were centrifuged (4°C) at 15 000 × g for 20 min. The resulting pellet was resuspended in the same solution. Protein was determined by the method of Lowry et al. [22]. Cell-free PLD assays were performed with 10-40 nmol of substrate (added as an aqueous sonicate) in a final volume of 0.2 ml of 20 mM Hepes/154 mM NaCl/1.25 mM EDTA/0.05% Triton X-100 (pH 7.0), in the presence or the absence of 2% ethanol, and protein as indicated. For radiolabeled membrane experiments, the 15000 × g particulate fractions were added to the assay system as the source of enzyme and substrate. The samples were incubated at 37"C with shaking, for 15-60 min. Reactions were terminated by adding 1.2 ml cold methanol containing 2% acetic acid, and lipids were extracted [19] and prepared for analysis as previously described [23].
Analysis of cellular metabolites and cell.free enzymatic products. Radiolabeled neutral lipids and phospholipids were resolved by TLC as previously described [23]. PEt was separated by TLC as previously described [5]. Water-soluble ',;H]choline-labeled products were isolated in the upper aqueous phase [19] and separated by TLC as described [24]. Desaturation of [3H]stearic acid by the cultured cells was determined by isolation of total radiolabeled cellular PC, as described above, followed by TLC resolution of methyl esters on silver nitrate-impregnated plates [25]. All labeled products, identified by their migration with commercial standards and visualized using iodine vapor, were quantitated by liquid scintillation spectrometry [5]. Results REF52 cells, transformed by SV40, exhibit many characteristics of tumor cells [17,18], including anchor-
244 age-independent growth and tumorigenicity. Our experiments were designed to compare PLD activity in the nontransformed and transformed fibroblasts as our earlier observations with these cells revealed that the transformed lines were deficient in PC hydrolysis when challenged with various agonists [23]. PLD activity was assessed in intact cells and via cell-free assay using exogenously added PC, prepared from both cell types, as well as membrane.bound PC.
PLD activity.response of intact REF52 and transformed celb to TPA and VP Labeling cells with myristic acid facilitates the study of PC metabolism in response to agonist treatment, since the labeling of inositol phospholipids is minimized [4,11], The distribution of radiolabei in total lipids from nontransformed and transformed cells, at" tot preincubation with either ['~H]myristie acid or [3H]glycerol, is shown in Table I. Although both cell types incorporated the majority of the ['~H]myristic acid into PC (68% in REF52; 42% in WT6 Ag6), the distribution of TAG-associated tritium in the two cell types differed. The data in Table I show that 5% and 37% of the total cellular 3H was associated with TAG TABLE 1 Distribution of tritium in celhdar lipids of REF52 and transformed WT6 Ag6 cells prclabeled with ['~H/my~tic acid or ['~H/glycerol Cells were labeled with I'~Hknyristic acid (I #.Ci/ml of 5% FBS medium) or l'~H]glycerol(5 ttCi/ml of 5% FBS medium) for 24 h, and total cellular lipids were extracted by the Bligh and Dyer [19]
procedure, Phospholipids and neutral lipids were resolved as previously described [23], and the lipids were analyzed for tritium as detailed in Materials and Methods, Data represent the mean of three cultures, Cellular
lipids
Incorporation ( ~ distributi°n°f total "~H) REF$~
{'~H]MA h
LPC Sph * PC
0,4±0,1 4.7±0,5 68,24.5,7
Inositol + serine PL PP_ MAG DAG FA * TAG Others c
3,4 ± 0.5 11,4± 1.4 !,0±0,2 0.4±0,1 0.1 ±0,01 4,7:t:0,9 5,7±0,3
W'r6 Ag6
I3HJG !,0±0,3 51.3 ±0.9 17,9:1:0.6 13.6±0.3 2.0±0.2 0.5±0,2
['~H]MA 0A±0,1 1.8±0,1 41.7± 1.6
!,5 +0.3 6.9:t:0,2 0.4:1:0,1 0.4±0.1 0,2 ± 0,01 1,0:t:0,1 36,8±5,2 12,3± 1,0 9.9:]:0.9
I'~H]G 2,1 ±0,1 53,9 + 0.8 t5,1 ± 1,1 16,5::1:0,4 6.3±0,4 0,6+0,1 3.3:J:0.5 1.5 ±0.1
* Sphingom~lin and free fatty acid radioactivity was lower than 0,01% in [3H~lycerol.labeled REF$2 and WT6 Ag6 cells, b MA, myristic acid; (3, glycerol; LPC, ]Ysophosphatidylcholine; Sph,
sphinlp)myelin: PC, phosphatidylcholine; PL, phospholipids; PP_, phosphatidy]ethanolamine; MAG, monoacylglycerol; DAG, diacylgb~rol; FA, fatty acid, TAG, triacylgbjcerol. c Denotes peP."ent of tritium at origin, solvent front, or unidentified on the TLC plate.
6
[
0
2
4
6 8 10 Time ( rain I Fig, 1. TPA-induced PLD activity in cultures of REF52 and transformed WT6 Ag5 cells. Cells were prelabeled with ['~Hlmyrisfic acid, equilibrated, and treated with TPA in medium containing 2% ethanol. Total cellular lipids were extracted and PEt was resolved and quantitatod as detailed in Materials and Methods. Data are the mean :l:[i.D. of three experiments at each time point. This experiment was performed two times with similar results. Here. and Fig. 2, the results are based on dpm of product formed per 500000 dpm of total cellular lipid radioactivity, e, REF52 cells; o, WT6 Ag6 cells,
in REF52 and WT6 Ag6 cells, respectively. In c 3. Enzyme preparations from both cell types displayed similar affinities for each type of PC. However, within a given enzyme preparation, PC derived from WT6 Ag6 cells was poorly hydrolyzed and accounted for 26 to 30% of the activity obtained with REF52 PC. When the substrates were analyzed with regard to degree of acyl group saturation [25], large differences were found. The results of argentation TLC of PC methyl esters demonstrated that 70% and 45% of the radioactivity was present as saturates in REF52 and WT6 Ag6 cells, respectively. Monoenoic species
80
6O
i 40
Cell-free PLD activity of normal at~d transformed cells The distribution of PLD total activity in the pellet and supernatant, prepared by 15 000 x g centrifugation of broken cells, was 88 and 12%, respectively, in both cell types. Using exogenous [3H]stearoyl-PC as substrate, the data in Fig. 3 show that PLD activity is, in contrast with results from intact cells, very similar in 15 000 × g preparations from both cell types. Further assessment of PLD activity was carried out using particulate preparations containing endogenous membrane-bound substrate and enzyme. When PLD activity was measured using this system, a difference in enzyme activity between normal and transformed cells was again readily apparent. The data in Fig. 4 show the influence of increased incubation time and substrate concentration on PLD activity in preparations from the two cell types. The formation of [3H]PEt as a function of time using equal amounts of protein was nearly
2O
0
0
.
i
20
40 Protein ( P8 )
60
80
Fig. 3. The influence of increased protein on cell-free PLD activity in particulate preparations from REF52 and WF6 Ag6 cells. The assays, incubated for 15 rain at 37°C, contained 20 mM Hepe,~ buffer (pH 7.0), 154 mM NaCI, 1.25 mM EDTA, (I,05% Triton X-100, 2~'/,. ethanol, 56 p.M [3H]stcaroyI-PC, and as enzyme source, the 150(g0 × g pellet (protein as indicated) from the respective cell line. REF52 enzyme contained 0,12 nmol endogenous PC per pg protein, and WT6 Ag6 enzyme contained 0.1 nmol endogenous PC per p.g protein. The substrate (PC) was obtained from REF52 cells. Assays were terminated by addition of lipid extraction solvents [19], and PEt was isolated and quantitated as described in Materials and Methods, Data points are the mean + S.D, of three separate enzyme assays, e, REF52 cells; o, WT6 Ag6 cells,
246 TABLE II
Influence of the source o f PC substrate on PLD activity measured in particulate preparations from REF52 and transformed WT6 Ag6 cells For cell-free assays, 0.2 ml volume contained exogenous 13H]stearoyI-PC (20 nmol), the 15000× g pellet from respective cell lines (50 ~tg protein) as enzyme, and other components as detailed in Materials and Methods. The enzyme preparation from REF52 cells and WT6 Ag6 cells contained approx. 6.0 and 5.0 nmol endogenous PC, respectively. After a 15 rain incubation at 37~C, reactions were terminated and [3H]PEt was quanfitated. Data represent mean ± S.D. from four experiments.
Enzyme source substrate source
PLD activity (p:nol PEt)
En~me of REF52 cells PC from REF52 cells PC from Wi~6 A86 cells
69.8 ± 3.5 18.2 ± 4.4
Enzyme of WT6 AS6 cells PC from REF$2 cells PC from WT6 AS6 cells
50.5:1:3,5 15,4 ± 2~8
nmol per assay) radiolabeled in the choline moiety as substrate (prepared from REF52 cells), and quantirated the release of [3H]choline as a measure of PLD activity. Results from these experiments showed that there were no differences in the liberation of [3H]choline from phosphatidyl['~H]choline in ethanolfree assays (REF52, 228.9 + 17.5 pmol/min per mg protein; WT6 Ag6, 215.7 + 20.4 pmol/min per mg protein) and ethanol-containing assays (REF52, 241.6 :i: 19.6 pmol/min per mg protein; WT6 Ag6, 211.5 :!: 23.5 pmol/min per mg protein), using 50/zg protein (15000 x g preparations) from either REF52 or WT6 Ag6 cells, as enzyme source. Discussion
accounted for 6% and 47% of the tritium in REF52 and WT6 A86 PC, respectively, whereas polyene radioactivity represented 18% and 5% in REF52 and WT6 AS6 cells. Our data are based on the formation of PEt as a measure of PLD activity. We therefore deemed it important to determine whether ethanol, per so, exerted any differential influence on PLD activity in the two cell types. To evaluate this we employed PC (40
As little is known about PC metabolism by PLD in normal and neoplastic cells, we employed the nontransformed rat embryo fibroblast line, REF52, and its SV40 transformed, tumorigenic counterpart, WT6 ~,g6 [17,18]. In earlier studies, before the enzymatic pathways (PLD versus PLC) were fully elucidated, we noted a correlation between decreased agonist-induced choline glycerophospholipid hydrolysis arid increased tumorigenicity in REF52 cells and the transformed variants [23]. In the present work we have investigated PC degradation by PLD, and characterized PC hydrolysis in cells treated with agonists and in cell-free preparations. When comparing PLD action in the two cell
A
B
20
8
le ,.~
--.
4
2
0
"
o
"
'
20
'
'
40
Time ( rain )
'
0
60
0
$0
10o
150
200
PC [ I~M J
Fig. 4. Cell-fr~ PLD activity in endogenous radiolabeled particulate preparations from REF$2 cells and transformed WT6 Ag6 cells. Cultured cells were prelab¢l~l with [3H]myristic acid, washed, equilibrated, and harvested to prepare the 15000× g radiolabaled particulate fractions (des~n'l~d in Materials and Methods). The assays contained buffer, NaCI, EDTA, Triton X-100, ethanol (see legend to Fig. 3), and the ndiolabeled I$000x Z paniculate fraction as source of both enzyme and substrate. The substrate concentration and radiospecific activity of PC in the labded 15000× g paniculate fraction were predetermined in random samples by extraction and isolation of PC followed by pbospbolipid phosphorus analysis 120]. (A) The influence of time on PEt formation (50 #g protein per assay). (B) The influence of substrate concentration on PEt formation. Data points represent the mean±S.D, from throe separate experiments. This experiment was performed two times with similar results, e, REF52 cells; o, WT6 Ag6 cells.
247 types, the agonist-inducible mechanisms as well as substrata type and availability are important factors to consider. The data from intact cells show that the formation of PEt via agonist-inducible pathways is higher in nontransformed cells as compared to the transformed cells. This conclusion is based on results from TPA- and VP-induced PEt formation in intact cell cultures (Figs. 1 and 2), and cell-free PLD assays utilizing particulate cellular preparations that contained endogenous enzyme and substrate (Fig. 4). As to the mechanism(s) underlying the differences in enzstme activity between the two cell types, insight can be gained from the experiments that were conducted to test substrata type and substrata availability. The results in Fig. 3 and Table II, using exogenous [3H]stearoyI-PC from REF52 cells, demonstrate that the formation of [3H]PEt (PLD activity) is very similar in particulate preparations from both cell types. For example, when enzyme from REF52 and WT6 Ag6 cells was presented with the same substrata, catalytic activity was similar. Conversely, when [3H]stearoyI-PC prepared from WT6 Ag6 cells was employed as substrate, enzymatic activity was poor (Table II). Overall, these d a t a suggest that PLD displays specificity for certain molecular species of PC. This poi,~t is strengthened by the data on the analysis of saturates/urnsaturates in PC derived from both cell types. In PC acyl groups from WT6 Ag6 cells, approx. 47% of the radioactivity was present as monoenes, whereas in REF52 cell PC 6% of the radioactivity was present in monoenes. In addition, polyenes (> 3 double bonds) accounted for 5% and 18% of the aliphatic radioactivity in PC of WT6 Ag6 and REF52 cells, respectively. Comparisons of the diradyl molecular species of precursors and products have been used to establish that PC is a source of phosphatidic acid and DAG in agonist-treated cells [26,27]. Those results and the present data indicate that certain subsets of PC are substrates for PLC a n d / o r PLD. Herein, our results further support the notion that mammalian PLD exerts a high degree of substrata specificity as regards the aliphatic portion of PC. Although there are difficulties inherent in interpreting enzymatic results based on radiolabeling of intact cells and membranes, our experiments to evaluate PLD action employed two agonists with dissimilar modes of action and two radiolabeled precursors that are incorporated into different portions of the PC molecule. These data demonstrate a deficit in the activity of PLD associated with the transformed derivative. The distribution of [3H]myristate in the two cell types was different, and this could account for differential PEt synthesis (Fig. 1). However, it is important to note that in cells radiolabeled with glycerol, wherein the phospholipid tritium profile was similar (Table I), the agonistinducible PLD was nevertheless different in the two
cell types (Fig. 2). When the data are viewed collectively, the results suggest that substrate specificity for PC molecular species plays a role in the action of agonist-inducible PLD and PLD in cell-free preparations from nontransformed and transformed fibroblasts. Whether the variances in PLD activity result from differences in membrane structure or are reflective of lipid composition, per se, is presently not known. From the standpoint of lipid composition, it is interesting to speculate that WT6 Ag6 cells are deficient in certain molecular species of PC. The role of r a s transformation in cell signaling pathways involving phospholipid breakdown has been the object of recent study [13-16]. The reports have shown that r a s transformation is accompanied by increased levels of cellular DAG, presumably by stimulation of PLC activity. Kundra and Dean [28] have reported that SV3T3 cells have reduced lysosomal enzyme activity compared to 3T3 cells. At the level of the ii~taet cell it is necessary to consider a myriad of reactions that may contribute to differences in PLD action in normal versus transformed cells. Among these variables one must include agonist type, receptor number, coupling mechanisms, and the interplay of numerous cellular factors, including protein kinase C isoenzymes [29]. Little is known regarding the influence of cellular transformation on PLD, a significant enzyme in signal transduction [3,4]. it is hoped that the results herein will set the stage for a more in-depth assessment of PC metabolism and the relationship of PLD to cell homeostasis.
Acknowledgments This work was supported by National Institutes of Health Grant CA-46530 and by Grant 2165 from The Council for Tobacco Research-USA, Inc. A portion of this research was carried out at The W. Alton Jones Cell Science Center in Lake Placid, New York. We wish to express our gratitude to Dr. Robert L. Wykle, Dr. Moseley Waite and Dr. Larry Daniel for their support, generosity and hospitality during the course of these studies. We thank Connie McArthm and April Comer for secretarial assistance.
References 1 2 3 4 5
Berridge, M.J, and lrvine, R.F. (1984) Nature 312, 315-32!, Nishizuka, Y. (1984) Science 225, 1365-1370, Exton, LH. (1990) J. Biol. Chem. 265, 1-4. L6ffelholz, K. (1989) Bioehem. Pharmacol. 38, 1543-1549, Huang, C. and Cabot, M.C, (1990) J. Biol. Chem. 265, 1485814863. 6 Dowries, C.P,, Hawkins, P.T. and Stephens, L. 0989) in Inositol Lipids in Cell Signalling (Michell, H,H., Drummond, A.H. and Downes, C,P,, ads.), pp. 3-38, Academic Press, Harcourt Brace, 7 Kroll, MH,, Zavoico, G,B. and Schafer, A,I. (1989) J. Cell. Physiol. 139, 558-564,
248 8 Bocckino, S.B., Blackmore, P.F., Wilson, P.B. and Exton, J.H. (1987) J. Biol. Chem. 262, 15309-15315. 9 Billah, M.M., Pai, J.-K., Mailman, TJ., Egan, R.W. and Siegel, M.I. (1989) J. Biol. Chem. 264, 9069-9076. 10 Agwu, D.E., McPhail, L.C., Chabot, M.C., Daniel, L.W., Wykle R.L. and McCall, C.E. (1989) J. Biol. Chem. 264, 1405-1413. !1 Cabot, M.C., Welsh, CJ., Cao, H-t. and Chabbott, H. (1988) FEBS Lett. 233, 153-157. 12 Qian, Z. and Drewes, L.R. (1989) J. Biol. Chem. 264, 2172021724. 13 Lacal, J.C., Moscat, J. and Aaronson, S.A. (1987) Nature 330, 269-272. 14 Slivka, S.R,, Godson, C. and lnsel, P.A. (1990) J. Cell, B,.'ochem. 42, 59-70, 15 Matyas, G.R. and Fishman, P,H, (1989) Cell. Signal. 1, 395-404. 16 Molar, J,, Cornet, M,, Diaz.Meco, M,T,, Larrodera, P., LopezAlamon, D, and Lopez.Barahona, M, (1989) Biochem, So¢, Trans. 17, 988-991, 17 M~lure, D,B,, Hightower, MJ, and Topp, W,C, (1982) in Cold Spring Harbor Conference on Cell Proliferation, Vol, 9, pp, 345-364, 18 MeClure, D,B,, Dermody, M, and Topp, W,C, (1984) in Cancer
19 20 21 22 23 24 25 26 27 28 29
Cell/the Transformed Phenotype, pp. 17-23, Cold Spring Harbor Laboratory, Cold Spring Harbor. Bligh, E.G. and Dyer, WJ. (1959) Can. J. Biochem. Physiol. 37, 911-917. Rouser, G., Siakotos, A.N. and Fleischer, A. (1966) Lipids 1, 85-86. Martin, T.W. (1988) Biochim. Biophys. Acta 962, 282-296. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, RJ. (1951) J. Biol. Chem. 193, 265-275. Cabot, M.C., Welsh, C.J., Zhang, Z-c., Cao, H-t., Chabbott, H. and Lebowitz, M. (1988) Biochim. Biophys. Acta 959, 46-5'7. Huang, C. and Cabot, M.C. (1990) J. Biol. Chem. 265, 1746817473. Blank, M.L., Lee, T-c., Piantadosi, C., Ishaq, K.S. and Snyder, F. (1976) Arch. Biochem. Biophys. 177, 317-322. Augert, G., Bocckino, S.B., Blackmore, P.F. and Exton, J.H. (1989) J. Biol. Chem. 264, 21689-21698. Pessin, M.S. and Raben, D.M. (1989) J. Biol. Chem. 264, 87298738. Kundra, V. and Dean, M.F. (~.990) Exp. Cell Res. 189, ~3-99. Nishizuka, Y. (1984) Nature 308, 693-698.
Biochimica et Biophysica Acta, 1127 (1992) 242-248 © 1992 Elsevier Science Publishers B.V. All rights reserved 0005-2760/92/$05.00
BBALIP 53968
Phospholipase D activity in nontransformed and transformed fibroblasts Chunfa Huang ~ and Myles C. Cabot Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Medical Center Boulevard, Winston.Salem, NC (USA)
(Received 28 February 1992)
Key words: Phospholipase D; Fibroblast; Transformed cell
R~t embryo fibroblasts (REF$2cells) and the simian virus 40 transformed derivative (WT6 Ag6) were employed to characterize phospholipase D (PLD) activity in normal and t~ansformed cells, in cells prelabeled with [3H]myristic acid or [3H]glycerol and treated with I2-O-tctrad¢canoylphorbol-13-acetate (TPA, 50 ng/ml medium) or vasopressin (VP, 100 ng/ml medium) in the presence of ethanol, the formation of labeled phosphatkiylethanol (PEt) was 3- to 5-fold higher in REF52 cells than in the transformed cells. The transphosphatidylation of phosphatidylcholine (PC) to PEt was further examined in cell-free assay systems. Results demonstrated that the formation of PEt in the cell-free assays was dependent on the mode of substrate presentation and the source of the PC. With endogenous membrane-bound substrate, the formation of [aH]myristoyi-PEt was 5-fold higher in homogenates derived from normal cells as compared to transformed cell homogenates. In experiments using exogenous labeled PC isolated from either REF52 or transformed cells as substrate, cell-free PLD activity differed greatly with regard to the source of the PC. The formation of PEt from REF52-derived PC was approx. 4-fold higher as compared to PEt formed with PC derived from the transformed cells, irrespective of enzyme source. The results demonstrate that PLD in intact nontransfotmed fibroblasts is activatable by TPA and VP to a greater extent than in the transformed counterpart. The results from cell-free assays suggest that PLD activity is more dependent on the type of PC substrate than on the source of the enzyme.
Introduction DAG, formed via phosphoinositide hydrolysis by PLC, is an important second messenger that promotes translocation and activation of protein kinase C [1,2], DAG is also a pivotal intermediate in cellular lipid metabolism. New evidence has recently shown that DAG in agonist-stimulated cells, can likewise be derived from PC by receptor-dependent activation of PLC and PLD [3-5]. PLD (EC 3.1.4.4) catalyzes the breakdown of the terminal phosphodiester bond of
C~dence to: M.C. Cabot, Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Medical Center Boulevard, Winston-Salem, NC 27157-1016, USA. t Visiting scientist from Xiamen University, Department of Biology, Xiamen, People's Republic of China. Abbz~iatiom: In the naming of the glyceropbospholipids, we have ~ distinguished between the type of aliphetic linkage to glycerol (es~r or ether). D&G, diacylllycerol; PLC, phospholipase C; PLD, p~,osO~lipzse 13;,PEt, phesphatidylethanol; REF$2, rat embryo ft. broblasts; SV4O, simian virus 40; TIPA, 12-O-tetradecanoylphorbol13-acetate; VP, vasopressin; BSA, bovine serum albumin; FIBS, fetal bovine serum; TLC, thin-layer chromatography; PC, phosphatidylcholine; TAG, triacylglycerol; PBS, phosphate-buffered saline.
glycerophospholipids to produce phosphatidic acid and the relevant bases. Phosphatidic acid, like DAG, is not only a key intermediate in recycling of the glycerol backbone [6], but may as well function as an element of cellular signal transduction [7]. In addition, phospha. tidic acid can be hydrolyzed to DAG by phosphatidic acid phosphatase. By measuring the formation of phosphatidic acid or PEt, several research groups have demonstrated the presence of agonist-stimulated PLD activity in nontransformed cultured animal cells and tissues [8-12]. Few studies have been undertaken to assess PLD activity in transformed cells, although some :~tudies have demonstrated increased DAG levels, via a mechanism attributed to PLC action in ms-transformed cells [1316]. In the present study rat embryo fibroblasts (REF52) and a SV-40 transformed derivative (WT6 Ag6) [17,18] were used to examine the relationship of cellular transformation to PLD activity. Herein, we have characterized for the first time, PLD activity in fibroblasts of nontransformed and transformed origin. The results reveal that, although agonist-induced PLD activity was higher in the nontransformed fibroblasts, when assessed by cell-free assay, enzyme activity was shown to be strongly dependent on the source/type of PC sub-
243 strate. It is hoped that these results will set the stage for future research on the role of PC and the action of PLD in cellular proliferation. Materials and Methods
Materials. [9,10(n)-3H]Myristic acid (39.3 Ci/mmol), [2-3H]glycerol (10 Ci/mmol) and [methyl-3H]choline chloride (86.7 Ci/mmol) were purchased from New England Nuclear (Boston, MA). [9,10(n)-3H]Stearic acid (60 Ci/mmol) was from American Radiolabeled Chemicals (St. Louis, MO). TPA was supplied by Chemicals for Cancer Research (Eden Prairie, MN). Cell culture media were purchased from Gibco (Ghithersburg, MD). [ArgS]VP and bovine serum albumin (BSA) were from Sigma (St. Louis, MO). Fetal bovine serum (FBS) was obtained from HyCIone (Logan, UT). Silica gel TLC plates were from Analtech (Newark, DE), and solvents were purchased from Burdick and Jackson (Muskegon, MI) and J.T. Baker (Phillipsburg, N J). Lipid standards for TLC were purchased from Avanti Polar Lipids (Alabaster, AL) and Nu Chek Prep (Elysian, MN). Cell culture, radiolabeling and treatment. The rat embryo-derived fibroblasts (REF52) and the SV40 transformed tumorigenic variant (WT6 Ag6), provided by Dr. D.B. McClure (Division of Molecular and Cellular Biology, Eli Lilly and Company, Indianapolis, IN), were cultured as previously described [17,18]. REF52 ceils were used at passage 8-18, and WT6 Ag6 cells were used at passage 15-18. Cells were labeled with [3H]myristic acid (1 /zCi/ml of 5% FBS medium) or [3H]glycerol (5/zCi/ml of 5% FBS medium) for 24 h. After removal of labeling medium, cultures were rinsed twice in serum-free medium containing 1 mg BSA/ml and further equilibrated by incubation in the same medium for 1 h ([3H]myristic acid-labeled cells) or 2 h ([3H]glycerol-labeled cells). Cells were treated with either TPA (50 ng/ml) or VP (100 ng/ml), as indicated in figures, in medium containing 2% ethanol. Experiments were terminated by extracting total cellular lipids using the Bligh and Dyer procedure [19], modified to contain 2% acetic acid in methanol. Radiolabeled PC substrates, subceUular fractionation, and cell.free reaction conditions. To prepare radiolabeled PC, REF52 cells were labeled with either [3H]stearic acid (20/~Ci/ml of 5% FBS medium) or [3H]choline (15/~Ci/ml of 5% FBS medium) for 48 h. Transformed cells were prelabeled with [3H]stearic acid (20 ~Ci/ml of 5% FBS medium) for 48 h. Cultures were then rinsed twice in serum-free medium (1 mg BSA/ml) and equilibrated for 2 h in the same medium. Total cellular lipids were extracted [19] and PC was resolved by preparative TLC using a solvent system containing c h l o r o f o r m / m e t h a n o l / a c e t i c acid/water (50: 27: 8: 2.5, v/v). The PC fraction was
eluted from the silica gel by a modified Bligh and Dyer procedure [19], in which a 5 ml volume of methanol/10% acetic acid/water (25: 8: 4, v/v) was added. After vortex mixing, the sample was set aside for 10 min and then 5 ml chloroform, 1.6 ml methanol, and 3.5 ml water were added. Samples were mixed and centrifuged, and the lower phase was filtered through glass wool-plugged pipets and evaporated with nitrogen. The radiospecific activity of substrates in labeled membranes and in labeled exogenous PC ([3H]stearoyl-PC from REF52, 18.3 t~Ci//~mol; phosphatidyl[3H]choline from REF52, 23.4 t~Ci/t~mol; [3H]stearoyl-PC from WT6 Ag6, 20.9 t~Ci/t~mol) was determined by phospholipid phosphorus analysis [20]. Subct:llular fractions were prepared essentially according to the procedure of Martin [21]. Briefly, cultures were washed three times with cold Ca 2+. and Mg2+-free PBS (pH 7.2). Cells were scraped in ice-cold 10 mM Hepes/154 mM NaCl/1 mM EDTA (pH 7.5) and sonicated for three 10-s intervals. The cell sonicates were centrifuged (4°C) at 15 000 × g for 20 min. The resulting pellet was resuspended in the same solution. Protein was determined by the method of Lowry et al. [22]. Cell-free PLD assays were performed with 10-40 nmol of substrate (added as an aqueous sonicate) in a final volume of 0.2 ml of 20 mM Hepes/154 mM NaCl/1.25 mM EDTA/0.05% Triton X-100 (pH 7.0), in the presence or the absence of 2% ethanol, and protein as indicated. For radiolabeled membrane experiments, the 15000 × g particulate fractions were added to the assay system as the source of enzyme and substrate. The samples were incubated at 37"C with shaking, for 15-60 min. Reactions were terminated by adding 1.2 ml cold methanol containing 2% acetic acid, and lipids were extracted [19] and prepared for analysis as previously described [23].
Analysis of cellular metabolites and cell.free enzymatic products. Radiolabeled neutral lipids and phospholipids were resolved by TLC as previously described [23]. PEt was separated by TLC as previously described [5]. Water-soluble ',;H]choline-labeled products were isolated in the upper aqueous phase [19] and separated by TLC as described [24]. Desaturation of [3H]stearic acid by the cultured cells was determined by isolation of total radiolabeled cellular PC, as described above, followed by TLC resolution of methyl esters on silver nitrate-impregnated plates [25]. All labeled products, identified by their migration with commercial standards and visualized using iodine vapor, were quantitated by liquid scintillation spectrometry [5]. Results REF52 cells, transformed by SV40, exhibit many characteristics of tumor cells [17,18], including anchor-
244 age-independent growth and tumorigenicity. Our experiments were designed to compare PLD activity in the nontransformed and transformed fibroblasts as our earlier observations with these cells revealed that the transformed lines were deficient in PC hydrolysis when challenged with various agonists [23]. PLD activity was assessed in intact cells and via cell-free assay using exogenously added PC, prepared from both cell types, as well as membrane.bound PC.
PLD activity.response of intact REF52 and transformed celb to TPA and VP Labeling cells with myristic acid facilitates the study of PC metabolism in response to agonist treatment, since the labeling of inositol phospholipids is minimized [4,11], The distribution of radiolabei in total lipids from nontransformed and transformed cells, at" tot preincubation with either ['~H]myristie acid or [3H]glycerol, is shown in Table I. Although both cell types incorporated the majority of the ['~H]myristic acid into PC (68% in REF52; 42% in WT6 Ag6), the distribution of TAG-associated tritium in the two cell types differed. The data in Table I show that 5% and 37% of the total cellular 3H was associated with TAG TABLE 1 Distribution of tritium in celhdar lipids of REF52 and transformed WT6 Ag6 cells prclabeled with ['~H/my~tic acid or ['~H/glycerol Cells were labeled with I'~Hknyristic acid (I #.Ci/ml of 5% FBS medium) or l'~H]glycerol(5 ttCi/ml of 5% FBS medium) for 24 h, and total cellular lipids were extracted by the Bligh and Dyer [19]
procedure, Phospholipids and neutral lipids were resolved as previously described [23], and the lipids were analyzed for tritium as detailed in Materials and Methods, Data represent the mean of three cultures, Cellular
lipids
Incorporation ( ~ distributi°n°f total "~H) REF$~
{'~H]MA h
LPC Sph * PC
0,4±0,1 4.7±0,5 68,24.5,7
Inositol + serine PL PP_ MAG DAG FA * TAG Others c
3,4 ± 0.5 11,4± 1.4 !,0±0,2 0.4±0,1 0.1 ±0,01 4,7:t:0,9 5,7±0,3
W'r6 Ag6
I3HJG !,0±0,3 51.3 ±0.9 17,9:1:0.6 13.6±0.3 2.0±0.2 0.5±0,2
['~H]MA 0A±0,1 1.8±0,1 41.7± 1.6
!,5 +0.3 6.9:t:0,2 0.4:1:0,1 0.4±0.1 0,2 ± 0,01 1,0:t:0,1 36,8±5,2 12,3± 1,0 9.9:]:0.9
I'~H]G 2,1 ±0,1 53,9 + 0.8 t5,1 ± 1,1 16,5::1:0,4 6.3±0,4 0,6+0,1 3.3:J:0.5 1.5 ±0.1
* Sphingom~lin and free fatty acid radioactivity was lower than 0,01% in [3H~lycerol.labeled REF$2 and WT6 Ag6 cells, b MA, myristic acid; (3, glycerol; LPC, ]Ysophosphatidylcholine; Sph,
sphinlp)myelin: PC, phosphatidylcholine; PL, phospholipids; PP_, phosphatidy]ethanolamine; MAG, monoacylglycerol; DAG, diacylgb~rol; FA, fatty acid, TAG, triacylgbjcerol. c Denotes peP."ent of tritium at origin, solvent front, or unidentified on the TLC plate.
6
[
0
2
4
6 8 10 Time ( rain I Fig, 1. TPA-induced PLD activity in cultures of REF52 and transformed WT6 Ag5 cells. Cells were prelabeled with ['~Hlmyrisfic acid, equilibrated, and treated with TPA in medium containing 2% ethanol. Total cellular lipids were extracted and PEt was resolved and quantitatod as detailed in Materials and Methods. Data are the mean :l:[i.D. of three experiments at each time point. This experiment was performed two times with similar results. Here. and Fig. 2, the results are based on dpm of product formed per 500000 dpm of total cellular lipid radioactivity, e, REF52 cells; o, WT6 Ag6 cells,
in REF52 and WT6 Ag6 cells, respectively. In c 3. Enzyme preparations from both cell types displayed similar affinities for each type of PC. However, within a given enzyme preparation, PC derived from WT6 Ag6 cells was poorly hydrolyzed and accounted for 26 to 30% of the activity obtained with REF52 PC. When the substrates were analyzed with regard to degree of acyl group saturation [25], large differences were found. The results of argentation TLC of PC methyl esters demonstrated that 70% and 45% of the radioactivity was present as saturates in REF52 and WT6 Ag6 cells, respectively. Monoenoic species
80
6O
i 40
Cell-free PLD activity of normal at~d transformed cells The distribution of PLD total activity in the pellet and supernatant, prepared by 15 000 x g centrifugation of broken cells, was 88 and 12%, respectively, in both cell types. Using exogenous [3H]stearoyl-PC as substrate, the data in Fig. 3 show that PLD activity is, in contrast with results from intact cells, very similar in 15 000 × g preparations from both cell types. Further assessment of PLD activity was carried out using particulate preparations containing endogenous membrane-bound substrate and enzyme. When PLD activity was measured using this system, a difference in enzyme activity between normal and transformed cells was again readily apparent. The data in Fig. 4 show the influence of increased incubation time and substrate concentration on PLD activity in preparations from the two cell types. The formation of [3H]PEt as a function of time using equal amounts of protein was nearly
2O
0
0
.
i
20
40 Protein ( P8 )
60
80
Fig. 3. The influence of increased protein on cell-free PLD activity in particulate preparations from REF52 and WF6 Ag6 cells. The assays, incubated for 15 rain at 37°C, contained 20 mM Hepe,~ buffer (pH 7.0), 154 mM NaCI, 1.25 mM EDTA, (I,05% Triton X-100, 2~'/,. ethanol, 56 p.M [3H]stcaroyI-PC, and as enzyme source, the 150(g0 × g pellet (protein as indicated) from the respective cell line. REF52 enzyme contained 0,12 nmol endogenous PC per pg protein, and WT6 Ag6 enzyme contained 0.1 nmol endogenous PC per p.g protein. The substrate (PC) was obtained from REF52 cells. Assays were terminated by addition of lipid extraction solvents [19], and PEt was isolated and quantitated as described in Materials and Methods, Data points are the mean + S.D, of three separate enzyme assays, e, REF52 cells; o, WT6 Ag6 cells,
246 TABLE II
Influence of the source o f PC substrate on PLD activity measured in particulate preparations from REF52 and transformed WT6 Ag6 cells For cell-free assays, 0.2 ml volume contained exogenous 13H]stearoyI-PC (20 nmol), the 15000× g pellet from respective cell lines (50 ~tg protein) as enzyme, and other components as detailed in Materials and Methods. The enzyme preparation from REF52 cells and WT6 Ag6 cells contained approx. 6.0 and 5.0 nmol endogenous PC, respectively. After a 15 rain incubation at 37~C, reactions were terminated and [3H]PEt was quanfitated. Data represent mean ± S.D. from four experiments.
Enzyme source substrate source
PLD activity (p:nol PEt)
En~me of REF52 cells PC from REF52 cells PC from Wi~6 A86 cells
69.8 ± 3.5 18.2 ± 4.4
Enzyme of WT6 AS6 cells PC from REF$2 cells PC from WT6 AS6 cells
50.5:1:3,5 15,4 ± 2~8
nmol per assay) radiolabeled in the choline moiety as substrate (prepared from REF52 cells), and quantirated the release of [3H]choline as a measure of PLD activity. Results from these experiments showed that there were no differences in the liberation of [3H]choline from phosphatidyl['~H]choline in ethanolfree assays (REF52, 228.9 + 17.5 pmol/min per mg protein; WT6 Ag6, 215.7 + 20.4 pmol/min per mg protein) and ethanol-containing assays (REF52, 241.6 :i: 19.6 pmol/min per mg protein; WT6 Ag6, 211.5 :!: 23.5 pmol/min per mg protein), using 50/zg protein (15000 x g preparations) from either REF52 or WT6 Ag6 cells, as enzyme source. Discussion
accounted for 6% and 47% of the tritium in REF52 and WT6 A86 PC, respectively, whereas polyene radioactivity represented 18% and 5% in REF52 and WT6 AS6 cells. Our data are based on the formation of PEt as a measure of PLD activity. We therefore deemed it important to determine whether ethanol, per so, exerted any differential influence on PLD activity in the two cell types. To evaluate this we employed PC (40
As little is known about PC metabolism by PLD in normal and neoplastic cells, we employed the nontransformed rat embryo fibroblast line, REF52, and its SV40 transformed, tumorigenic counterpart, WT6 ~,g6 [17,18]. In earlier studies, before the enzymatic pathways (PLD versus PLC) were fully elucidated, we noted a correlation between decreased agonist-induced choline glycerophospholipid hydrolysis arid increased tumorigenicity in REF52 cells and the transformed variants [23]. In the present work we have investigated PC degradation by PLD, and characterized PC hydrolysis in cells treated with agonists and in cell-free preparations. When comparing PLD action in the two cell
A
B
20
8
le ,.~
--.
4
2
0
"
o
"
'
20
'
'
40
Time ( rain )
'
0
60
0
$0
10o
150
200
PC [ I~M J
Fig. 4. Cell-fr~ PLD activity in endogenous radiolabeled particulate preparations from REF$2 cells and transformed WT6 Ag6 cells. Cultured cells were prelab¢l~l with [3H]myristic acid, washed, equilibrated, and harvested to prepare the 15000× g radiolabaled particulate fractions (des~n'l~d in Materials and Methods). The assays contained buffer, NaCI, EDTA, Triton X-100, ethanol (see legend to Fig. 3), and the ndiolabeled I$000x Z paniculate fraction as source of both enzyme and substrate. The substrate concentration and radiospecific activity of PC in the labded 15000× g paniculate fraction were predetermined in random samples by extraction and isolation of PC followed by pbospbolipid phosphorus analysis 120]. (A) The influence of time on PEt formation (50 #g protein per assay). (B) The influence of substrate concentration on PEt formation. Data points represent the mean±S.D, from throe separate experiments. This experiment was performed two times with similar results, e, REF52 cells; o, WT6 Ag6 cells.
247 types, the agonist-inducible mechanisms as well as substrata type and availability are important factors to consider. The data from intact cells show that the formation of PEt via agonist-inducible pathways is higher in nontransformed cells as compared to the transformed cells. This conclusion is based on results from TPA- and VP-induced PEt formation in intact cell cultures (Figs. 1 and 2), and cell-free PLD assays utilizing particulate cellular preparations that contained endogenous enzyme and substrate (Fig. 4). As to the mechanism(s) underlying the differences in enzstme activity between the two cell types, insight can be gained from the experiments that were conducted to test substrata type and substrata availability. The results in Fig. 3 and Table II, using exogenous [3H]stearoyI-PC from REF52 cells, demonstrate that the formation of [3H]PEt (PLD activity) is very similar in particulate preparations from both cell types. For example, when enzyme from REF52 and WT6 Ag6 cells was presented with the same substrata, catalytic activity was similar. Conversely, when [3H]stearoyI-PC prepared from WT6 Ag6 cells was employed as substrate, enzymatic activity was poor (Table II). Overall, these d a t a suggest that PLD displays specificity for certain molecular species of PC. This poi,~t is strengthened by the data on the analysis of saturates/urnsaturates in PC derived from both cell types. In PC acyl groups from WT6 Ag6 cells, approx. 47% of the radioactivity was present as monoenes, whereas in REF52 cell PC 6% of the radioactivity was present in monoenes. In addition, polyenes (> 3 double bonds) accounted for 5% and 18% of the aliphatic radioactivity in PC of WT6 Ag6 and REF52 cells, respectively. Comparisons of the diradyl molecular species of precursors and products have been used to establish that PC is a source of phosphatidic acid and DAG in agonist-treated cells [26,27]. Those results and the present data indicate that certain subsets of PC are substrates for PLC a n d / o r PLD. Herein, our results further support the notion that mammalian PLD exerts a high degree of substrata specificity as regards the aliphatic portion of PC. Although there are difficulties inherent in interpreting enzymatic results based on radiolabeling of intact cells and membranes, our experiments to evaluate PLD action employed two agonists with dissimilar modes of action and two radiolabeled precursors that are incorporated into different portions of the PC molecule. These data demonstrate a deficit in the activity of PLD associated with the transformed derivative. The distribution of [3H]myristate in the two cell types was different, and this could account for differential PEt synthesis (Fig. 1). However, it is important to note that in cells radiolabeled with glycerol, wherein the phospholipid tritium profile was similar (Table I), the agonistinducible PLD was nevertheless different in the two
cell types (Fig. 2). When the data are viewed collectively, the results suggest that substrate specificity for PC molecular species plays a role in the action of agonist-inducible PLD and PLD in cell-free preparations from nontransformed and transformed fibroblasts. Whether the variances in PLD activity result from differences in membrane structure or are reflective of lipid composition, per se, is presently not known. From the standpoint of lipid composition, it is interesting to speculate that WT6 Ag6 cells are deficient in certain molecular species of PC. The role of r a s transformation in cell signaling pathways involving phospholipid breakdown has been the object of recent study [13-16]. The reports have shown that r a s transformation is accompanied by increased levels of cellular DAG, presumably by stimulation of PLC activity. Kundra and Dean [28] have reported that SV3T3 cells have reduced lysosomal enzyme activity compared to 3T3 cells. At the level of the ii~taet cell it is necessary to consider a myriad of reactions that may contribute to differences in PLD action in normal versus transformed cells. Among these variables one must include agonist type, receptor number, coupling mechanisms, and the interplay of numerous cellular factors, including protein kinase C isoenzymes [29]. Little is known regarding the influence of cellular transformation on PLD, a significant enzyme in signal transduction [3,4]. it is hoped that the results herein will set the stage for a more in-depth assessment of PC metabolism and the relationship of PLD to cell homeostasis.
Acknowledgments This work was supported by National Institutes of Health Grant CA-46530 and by Grant 2165 from The Council for Tobacco Research-USA, Inc. A portion of this research was carried out at The W. Alton Jones Cell Science Center in Lake Placid, New York. We wish to express our gratitude to Dr. Robert L. Wykle, Dr. Moseley Waite and Dr. Larry Daniel for their support, generosity and hospitality during the course of these studies. We thank Connie McArthm and April Comer for secretarial assistance.
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