(CANCER RESEARCH 43, 5564-5569, November 1983]

Effects of a Tumor Promoter on Phospholipid Metabolism in HeLa Cells1

Graeme R. Guy and Andrew W. Murray2

School of Biological Sciences, Flinders University, Bedford Park, South Australia 5042

ABSTRACT

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate(TPA) caused a marked stimulation of inorganic [32P]orthophos-

phate incorporation into HeLa-cell phosphatidylcholine (PC),phosphatidylethanolamine (PE), and lysophosphatidylethanola-mine. The increased incorporation of inorganic [32P]orthophos-

phate into PE and lysophosphatidylethanolamine in the presenceof TPA was not associated with an increase in PE synthesis asdetected by the incorporation of [3H]serine or [3H]ethanolamine.

The PC-specific exchange protein from beef liver was used toinsert PC labeled with [3H]choline, inorganic [32P]orthophos-phate, or [14C]arachidonic acid plus [3H]palmitic acid into the

outer monolayer of intact HeLa cell membranes. Radioactivityfrom the latter two compounds was rapidly incorporated into PEand lysophosphatidylethanolamine; the incorporation was stimulated by TPA. It was concluded that TPA stimulated the formation of PE by base exchange between ethanolamine and PC.

INTRODUCTION

Recent studies have demonstrated that the phorbol estertumor promoters cause rapid changes in PC3 turnover in cultured

mammalian cells (7, 8, 18). Both increased incorporation of[3H]choline into PC (8, 18) and increased release of radioactivityinto the medium from cells prelabeled with [3H]choline have been

reported (8,18). The current evidence suggests that the primaryreaction activated by promoters is a PC-specific phospholipase

C resulting in the production of DG and phosphocholine (7, 8,18). Grove and Schimmel (7) have provided evidence that aportion of the DG is converted into PA, which, in turn, is aprecursor for PI biosynthesis.

Perturbation of PC turnover by promoters occurs rapidly (8,18) and is a prime candidate for the initial event triggered bypromoters. For HeLa cells, at least, it has been established thatthe enhanced turnover is closely coupled to receptor occupancy,and removal of the promoter by washing causes a rapid returnto control rates (9).

In the present paper, 2 approaches have been used to explorefurther the effects of tumor promoters on HeLa cell phospholipidmetabolism: (a) overall patterns of 32P,incorporation into individ

ual phospholipids have been analyzed; (b) a technique has beendeveloped to insert PC molecules, prelabeled in a defined way,into intact HeLa cell membranes; the effect of TPA on the

1This study was supported by the Australian Research Grants Committee, the

National Health and Medical Research Council, and the Universities of SouthAustralia Anti-Cancer Foundation.

2To whom requests for reprints should be addressed.3The abbreviations used are. PC. phosphatidylcholine; DG, 1,2-diacylglycerol;

PA, phosphatidic acid; PI, phosphatidylinositol; TPA, 12-O-tetradecanoylphorbol-13-acetate; PE, phosphatidylethanolamine; PS, phosphatidylserine; lyso-PC, lyso-phosphatidylcholine; lyso-PE, lysophosphatidylethanolamine; MEM, Eagle's minimal essential medium; PBS. Dulbecco's phosphate-buffered saline (137 mw

NaCI:2.7 rriM KCI:0.5 mM MgCI2:0.9 mM CaCI2:8 mw Na2HPO4:1.5 mM KH2PO,, pH7.2); DMSO, dimethyl sulfoxide; lyso-PI, lysophosphatidylinositol.

Received October 8, 1982; accepted August 11, 1983.

redistribution of the labeled PC has then been determined.Insertion of the PC was accomplished using a PC-specific ex

change protein from beef liver, which catalyzes the exchange ofPC between liposomes and a variety of membrane preparations(4,12,15).

MATERIALS AND METHODS

Materials. TPA was obtained from P-L Biochemicals, Inc., Milwaukee,Wis. Phospholipase C (Clostridium perfringens), phospholipase A2 (Cro-talus adamanteus), PC, PE, PS, PI, PA, sphingomyelin, lyso-PC, lyso-PE,

and DG were all obtained from the Sigma Chemical Co., St. Louis, Mo.The Ca2* ionophore A23187 was a gift from Eli Lilly and Co., Indianapolis,

Ind. MEM and fetal calf serum were obtained from Commonwealth SerumLaboratories, Melbourne, Australia. Supplemented MEM contained a50% increase in essential amino acids, glutamine, and vitamins, a 100%increase in nonessential amino acids, and 1 mw sodium pyruvate.

[meffiy/-3H]Choline chloride (specific activity, 77 Ci/mmol), [9,10-3H]-palmitic acid (specific activity, 15.2 Ci/mmol), myo[2-3Hjinositol (specificactivity, 3.9 Ci/mmol), [1-3H]ethan-1-ol-2-amine hydrochloride (specificactivity, 8.8 Ci/mmol), L-[3-3H[serine (specific activity, 28 Ci/mmol), [1-'"CJarachidonic acid (specific activity, 58.4 mCi/mmol), and 32Pi were

obtained from the Radiochemical Centre, Amersham, England.HeLa cells were maintained in 35- or 60-mm plastic dishes (Lux

Scientific Corp., Newbury Park, Calif.) in MEM supplemented with 10%fetal calf serum and antibiotics (penicillin, 100 ID/ml; streptomycin, 100A/g/ml; complete medium). Incubations were carried out at 37° in a

humidified atmosphere of 5% CO2 in air, and cultures were used at celldensities of 1.5 to 2 x 106 cells/35-mm dish or 3.8 to 4.3 x 106 cells/

60-mm dish.

Polyethyleneimine cellulose plates (glass backed) and Silica Gel 60plates (aluminum backed) were from E. Merck, Darmstadt, Germany.Autoradiographs were carried out using Kodak X-Omat RP film.

PC-specific exchange protein was purified from beef liver essentially

as described (12) and used after the carboxymethylcellulose step. Theenzyme was stored at -20° in 50% glycerol. Exchange protein activitywas assayed by measuring radioactivity transferred from [3H]PC-labeled

multilamellar vesicles to freshly prepared liposomes (4).Methods. The following procedure was used to measure the incor

poration of precursors into phospholipids. Complete medium was removed from culture dishes, and the cells were washed twice with MEM.MEM (3 ml), together with either [3H]chlorine (0.3 Ci/ml), [3H]ethanol-amine (1 ^Ci/ml), [3H]serine (1 MCi/ml), [3H]inositol (1 nCi/ml), or 32R (20

AiCi/ml), was added, and incubations were carried out for the appropriatetime. TPA was added as a solution in DMSO; control incubationscontained DMSO alone (final solvent concentration, 0.1%). The mediumwas aspirated, and the cells were washed with ice-cold PBS prior to

extraction as described by Wertz and Mueller (21). The extracted phospholipids were dried in air, dissolved in 100 ¿ilof chlorofornrmethanol(2:1, v/v), and stored on ice under a N2 atmosphere. Phospholipids wereresolved by 2-dimensional chromatography on silica gel plates. Control

experiments established that the phospholipid patterns obtained fromair-dried samples were identical to those obtained when samples weredried in a stream of nitrogen. A solvent system of chloro-

form:methanol:ammonia (65:25:5, v/v) was used in the first direction,after which the plates were air dried for 10 min and oven dried (50-60°)

for 30 min. The plates were developed in the second direction with asolvent system of chloroform:acetone:methanol:acetic acid:H2O (30:

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Tumor Promoters and Phospholipid Metabolism

40:10:10:5, v/v). After chromatography, phospholipids were located byautoradiography or by staining with iodine vapor. Areas of silica gelcontaining phospholipid were scraped into vials, and radioactivity wasmeasured using a Triton:xylene scintillation fluid (1).

The various phospholipids were identified using authentic standardsand by determination of labeling patterns after incubation with [3H]-choline, [3H]inositol, [3H]serine, or [3H]ethanolamine. The identity of lyso-derivatives was confirmed by incubation of 3H- or 32Prlabeled PC, PI, or

PE (isolated from chromatograms) with phospholipase A2 and redeveloping the 2-dimensional chromatograms with appropriate unlabeledstandards. The 2-dimensional system described above did not com

pletely resolve PI and PS. A separate solvent (17) was used to separatethese 2 phospholipids.

Incorporation of 32P¡into HeLa-cell ATP was measured as follows.Cells were incubated in MEM containing 32P,(10 MCi/ml). At appropriate

times, the medium was removed, the cells were washed with ice-coldPBS (3x2 ml), and nucleotides were extracted with cold 5% trichloro-

acetic acid. After removal of the trichloroacetic acid by extraction withwater-saturated ether, an aliquot of the acid-soluble fraction was frac

tionated by chromatography on polyethyleneimine plates (14). Radioactivity associated with ATP was determined by scintillation counting.

Labeled PC was prepared by incubating HeLa cell cultures with[3H]choline (2 pCi/ml), 32P,(2 ^Ci/ml), or ["Cjarachidonic acid (2 MCi/ml)plus [3H]palmitic acid (1.3 /¿Ci/ml)for 2 to 3 days. Phospholipids were

then extracted, and PC was separated using 2-dimensional chromatog

raphy on silica gel plates.The transfer of labeled PC to cultured HeLa cells was carried out as

follows. Multilamellar vesicles were prepared (4) from each labeled PCspecies. Vesicles (approximately 2.5 x 10s cpm; 12.5 nmol of PC),exchange protein (0.1 mg protein), and 2 ml of Ca2+/Mg2+-free PBS wereadded to each 60-mm dish. The dishes were incubated for 2 hr at 37°

in a humidified atmosphere. The cells were washed with MEM (4x1ml), and incubations were continued in the presence of TPA (10~7 M) or

DMSO (0.1%) for various times. The phospholipids were then extractedand separated by 2-dimensional chromatography as described above.

RESULTS

We have shown previously that TPA causes a rapid stimulationof the release of radioactive choline and phosphocholine fromHeLa cells prelabeled with [3H]choline (8). These products are

derived from the PC pool and are a consequence of phospholipase C action (8). Release is followed by a TPA-enhanced rateof PC synthesis, as detected by the incorporation of [3H]choline

into phospholipid.A series of experiments was initially carried out to determine

the effect of TPA on the overall patterns of 32P,incorporation into

individual HeLa phospholipids. An example of an autoradiographshowing the separation of the individual phospholipids is shownin Fig. 1. The criteria used for identification of the separatedcompounds are summarized in "Materials and Methods." Not all

of the radioactive compounds were identified on these chromatograms, particularly the more polar derivatives with low mobilitiesin both solvents. This low-mobility group includes both phospha-tidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphos-phate. In these experiments, 32P,was added at Time 0, and it

was necessary to determine whether TPA caused any changein the incorporation of label into ATP which could lead to artifac-

tual changes in phospholipid labeling. A continuous increase in32P,labeling of ATP was observed over 120 min, and 1(T7 M TPA

caused a 30 to 43% increase in labeling (Chart 1). Although thisdata does not give a true estimate of ATP specific activity, weconclude that any minor TPA-induced increases in 32P,phospho

lipid labeling (up to about 1.5-fold) may result from this source.

0PA •*PQ PA

LPA

PI

LPE<

LPA

PC L

Fig. 1. Autoradiograph of 2-dimensional thin-layer chromatography of cell phospholipids extracted from DMSO and TPA-treated cells. Samples were processedas described in "Materials and Methods" 2 hr after incubation with DMSO or 10~7

M TPA. After 2-dimensional chromatography, phospholipids were visualized byradioautography; the direction of the second development is indicated with anarrow. LPA, LPE, and LPC, the lyso- derivatives of PA, PE, and PC, respectively;PG, phosphatidylglycerol. Left, 2-hr exposure to DMSO; right, 2-hr exposure toKT'MTPA.

60

TIME (Mm)Chart 1. Effect of TPA on the incorporation of Å“P, into ATP. Incubations were

carried out in the absence (•)or presence (O) of 10~7 M TPA. Each point is the

mean of triplicate determinations. Bars, S.E.

The results from a typical 32P, incorporation experiment are

shown in Table 1; similar data have been obtained in 10 separateexperiments. 32P¡incorporation into PC was rapidly stimulated

by TPA. It is likely that there was some increase in the labelingof PI and PA, particularly at the later time points, but thestimulation was not marked. This is also indicated by the observation that TPA caused a delayed and relatively minor stimulationof [3H]inositol incorporation into phospholipid (Chart 2).

A surprising result was that TPA caused a rapid and markedincrease in the 32P¡labeling of PE and lyso-PE (Table 1). However,

in contrast to PC (8), this was not accompanied by enhancedphospholipid synthesis, as detected by the incorporation of[3H]ethanolamine (Table 2). Another mechanism for the 32P,labeling of PE and lyso-PE is the decarboxylation of [32P,]PS. PS

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G. R. Guy and A. W. Murray

Table 1Effect of TPAon the incorporation of aPt into HeLacell phospholipids

Determinations were carried out as described in "Materials and Methods" afterincubation for the appropriate time in the presenceof DMSO or 10~7MTPA.

Incubation time

(min)306090120Radioactivity

(cpm/dish)TreatmentDMSO

TPADMSO

TPADMSO

TPADMSO

TPAPC188

794726

2,6301,062

4,5021,492

9,462Lyso-PC124

364193

794305

1,1554291,521PI794

1,1121,663

2,1762,293

2,4533,224

5,704PA410

606905

1,1351,237

2,3231,738

5,043PE304

1,194842

4,9901,130

9,7061,587

18,458Lyso-PE480

1,2951,059

6,4531,614

9,5302,272

18,679

o 10-

= 8-

C 6

o.

20 40 60 80

TIME (Min)

100 120

Chart 2. Effect of TPA on the incorporation of [3H]inositol into HeLa-cellphospholipids. Incubations were carried out in the absence (•)or presence (O) of 10~7

MTPA. Eachpoint is the mean of triplicate determinations.Bars, S.E.

is not synthesized de novo in animal cells (13,19) and is probablysynthesized by base exchange enzymes. As shown in Table 3,[3H]serine is incorporated rapidly into PS and PE, with lower

incorporation into other phospholipids. TPA caused a markeddecrease in [3H]serine incorporation into PE and a smaller de

crease in lyso-PE labeling (Table 3). This decrease was not dueto TPA inhibition of serine uptake into the acid-soluble pool (data

not shown). It is noteworthy that the ratio of incorporation in theabsence and presence of TPA is higher with PS (2.3 to 3.6) thanwith PE (1.1 to 1.5). Thus, in the presence of TPA, a higherproportion of the labeled PS pool is converted into PE. Theconclusion from Tables 2 and 3 is that the dramatic increase in32P, labeling of PE and lyso-PE induced by TPA cannot be

explained by gross changes in PE synthesis de novo or from PSdecarboxylation.

We have shown previously (8) that enhanced turnover of PCin HeLa cells is also induced by incubating cells with exogenousC. perfringens phospholipase C, or with the Ca2+ ionophore

A23187. As shown in Table 4, these agents also stimulated theincorporation of 32P,into PC, PI, lyso-PI, and PA; these results

were reproducible in 3 separate experiments. Stimulation of lyso-

PI labeling by A23187 was particularly marked and presumablyreflects a Ca2+ stimulation of a phospholipase A2 (3). However,it is noteworthy that neither the phospholipase C nor the Ca2+ionophore stimulated the 32P,labeling of PE or lyso-PE.

One possible mechanism for a selective TPA stimulation of the

incorporation of 32Pibut not of [3H]ethanolamine into PE would

be a base exchange reaction between PC and ethanolamine (13,19). In the presence of TPA, 32Piincorporation into PC is stimu

lated, and this would lead to increased label in PE if baseexchange occurred. Consequently, in an attempt to determinewhether such exchange reactions occurred, experiments werecarried out in which prelabeled PC was inserted into the outermonolayer of HeLa cell membranes. It was hoped that thisapproach would enable a distinction to be made between phos-

pholipid labeling by base exchange and by de novo biosyntheticpathways.

Preliminary experiments established that beef liver exchangeprotein catalyzed the efficient transfer of [3H]choline-labeled PC

into HeLa cell membranes. In a typical experiment, in whichvesicles containing approximately 2.5 x 105 cpm in PC wereincubated with HeLa cells as described in "Materials and Methods," about 6.5 x 104 cpm were inserted into the membranes

after a 2-hr incubation. Less than 1% of this incorporation was

observed in the absence of added exchange protein. Approximately 95% of the radioactivity was associated with PC, 4%with lyso-PC, and 1% with sphingomyelin (data not shown). As

expected, the majority of the label was initially in the outermonolayer of the membrane, as indicated by its susceptibility toexogenous phospholipase attack. Thus, incubation of prelabeledcells with exogenous phospholipase C (1 unit/ml) resulted in theloss of 86% of the radioactivity associated with PC over a 3-hrperiod. In addition, the incorporated PC was susceptible toincreased breakdown in the presence of TPA, as detected bythe release of radioactivity into the medium (Chart 3). In other

Table 2Effect of TPAon incorporation of [3H¡ethanolamineinto HeLa cell phospholipids

Determinationswere carried out as described in "Materials and Methods" afterincubation for the appropriate time in the presence of DMSO or 10~7MTPA.

Incubationtime(min)306090Radioactivity

(cpm/dishxTreatmentDMSOTPADMSOTPADMSOTPAPE385a290470560633520Lyso-PE154105220219394252PC3.22.26.07.05.84.310-3)Lyso-PC2.82.06.16.512.711.9

Mean of duplicate determinations.

Table 3Effect of TPAon incorporation of [3H]serine into HeLa-cellphospholipids

Determinationswere carried out as described in "Materials and Methods" afterincubation for the appropriate time in the absence or presence of 10~7MTPA.

Incubation time

(min)306090120Radioactivity

(cpm/dish)Treat

mentDMSOTPADMSOTPADMSOTPADMSOTPAPS3,914"1,4005,1741,4326.5472,1217,4643,272PE5453941,0819961,6711,1752,4721,650Lyso-PE115109139126169142205158PC155181269221270311378342Lyso-PC187139159120215127203171Sphingomyelin244185333261418403765705

a Mean of duplicate determinations.

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Tumor Promoters and Phospholipid Metabolism

Table 4Effecf of TPA, A23187, and phospholipase C on XP¡incorporation into HeLa cell phospholipids

Determinations were carried out as described in "Materials and Methods" after incubation for 2 hr in thepresence of DMSO, 10"7 M TPA, Clostridium perfringens phospholipase C (0.15 unit/ml), or A23187 (2 >IM).

Radioactivity (cpm/dish)

Treatment PC Lyso-PC PI Lyso-PI PE Lyso-PE PA

DMSOTPAPhospholipase

CA231871,3567,9445,2873,3064071,7821,5235982,9574,8444,7986,8525018401,1645,0611,55817,1201,3112,0812,08916,0631,2501,5481,6874,8874,9504,600

oX

o

i•

.S•o(Off O

DMSO

20 40 60 80 100 120

Time ( min )

Charts. The effect of TPA on the release of radioactivity from HeLa cellscontaining PC prelabeled with [3H]choline. Cells were incubated with PC exchangeprotein and vesicles containing [3H]choline-labeled PC as described in "Materialsand Methods." The cells were washed and incubated for varying times in thepresence of DMSO (O) or 10~7 M TPA (•),and radioactivity released into the

medium was measured.

studies, we have shown that this release is a consequence ofphospholipase C action (8).

Experiments were next carried out with HeLa cells which hadincorporated PC labeled with 32P((Table 5). It is clear that there

was rapid labeling of both PE and lyso-PE and that this was

stimulated by TPA. We propose that this result was a consequence of an increased rate of base exchange between etha-nolamine and PC. The alternative is that the inserted [32Pi]PCwas degraded to P;, leading to [32P¡]ATPformation and labeling

of PE via de novo biosynthetic pathways. However, we believethis to be unlikely, because the earlier experiments indicated that32Piwas rapidly incorporated into both PI and PA (Table 1). Only

low levels of radioactivity were associated with these compoundsin the experiments summarized in Table 5. However, it is obviousthat the TPA stimulation of PE and lyso-PE labeling from prela

beled PC (Table 5) was much less than that observed from the32P, incorporation data (Table 1). This is presumably at leastpartially because TPA also stimulates the incorporation of 32P(

into PC.The data in Table 5 also indicate that TPA stimulated the

accumulation of label in lyso-PC, presumably by activation ofphospholipase A2, as reported previously (2, 6). As predictedfrom the data in Chart 3 and from earlier work (8), TPA alsostimulated the release of radioactivity into the medium.

The possibility that HeLa cells catalyze a rapid base exchangereaction between ethanolamine and PC, which is stimulated byTPA, is supported by the data in Table 6. In these experiments,PC labeled with both [3H]palmitic acid and [14C]arachidonic acid

was inserted into HeLa cell membranes. There was a rapidincorporation of both 14C and 3H label into PE and of 3H label

into lyso-PE; both incorporations were stimulated by TPA. This

result suggests that direct transfer of both fatty acid moietiesfrom PC to PE occurs via a base exchange enzyme. There isevidence also for an enrichment of PI and possibly other phospholipids with arachidonic acid (14Clabel), which is unaffected byTPA. This could either result from the reincorporation of [14C]-

arachidonic acid released by phospholipase A2 action or from a"shuttle" involving the transfer of arachidonic acid from PC to

lysophosphatides catalyzed by lysophosphatide acyltransferase(11,16,20).

Accumulation of 14Cand 3H label also occurred in PS, and this

is presumably a consequence of serine exchange with either PEor PC. This reaction was slightly inhibited by TPA. In animal cells,PS is believed to be synthesized by base exchange betweenserine and other phospholipids rather than by de novo biosynthesis (13, 19). In the experiments in which 32P, incorporation

was measured, only low levels of radioactivity were associatedwith PS, even at the longer incubation times (data not shown).Consequently, it does seem that the PC pools labeled via denovo synthesis and exchange with exogenous PC are not identical. Although both are perturbed by TPA, the pool labeled withexogenous PC seems relatively more available for a subsequentexchange reaction with serine than that labeled with 32P,.

DISCUSSION

The present results confirm that a major and early responseof HeLa cells to the phorbol ester tumor promoters is an enhanced rate of PC turnover. Some delayed perturbation of PImetabolism is also induced, but this may largely be an indirectconsequence of the close interrelationship between the variousphospholipid pools. Evidence for such an interrelationship hasbeen reported from several laboratories (5, 10). For example,although adrenocorticotropic hormone is thought primarily toactivate the PI cycle, it also causes a marked stimulation of PCand PE metabolism (5). Similarly, platelet-derived growth factor

enhances PI turnover in 3T3 cells but also increased cholineincorporation into PC (10). However, it should be stressed thatthe analysis of PI metabolism in HeLa cells is not yet complete,as the fractionation methods used in the present study did notseparate the polyphosphoinositides phosphatidylinositol-4-phos-phate and phosphatidylinositol-4,5-bisphosphate. A rapid effect

of promoters on the hydrolysis of these derivatives has, therefore, not been ruled out.

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Table 5Effect of TPA on the redistribution of ^P-labeled PC in HeLa cell membranes

Cells were incubated with PC exchange protein in vesicles containing "Pi-labeled PC as described in"Materials and Methods." The cells were washed as described and incubated for 1 or 2 hr in the presence ofDMSO or TPA (10~7 M). Phospholipids were separated as described in "Materials and Methods."

Radioactivity (cpm/dish x 10"3)

CompoundPC

Lyso-PCPELyso-PE

PIPSPATimeO37.3

±1.1*

0.71 ±0.031.9 ±0.070.8 ±0.040.26 ±0.030.75 ±0.040.23 ±0.04DMSO

(1hr)32.5

±0.50.99 ±0.052.7 ±0.041 ±0.080.30 ±0.011.2 ±0.040.32 ±0.02TPA

(1hr)27

±0.83.1 ±0.115.6 ±0.32 ±0.090.31 ±0.040.92 ±0.080.36 ±0.04DMSO

(2hr)29.8

±0.62.8 ±0.284.1 ±0.181.4 ±0.140.36 ±0.021.7 ±0.120.38 ±0.01TPA

(2hr)22.6

±14.8 ±0.448 ±0.242.3 ±0.200.39 ±0.061.2 ±0.030.4 ±0.05

Released to medium 0.60 ±0.03 1.7 ±0.07 0.91 ±0.05 3.3 ±0.08" Mean ±S.E. of 3 replicate determinations.

Table 6Effect of TPA on the redistribution of [i4C]arachidonic acid and [3H]palmitic acid-

labeled PC in HeLa-cell membranes

Cells were incubated with PC exchange protein and vesicles containing PClabeled with [3H]palmitic acid and [14C]arachidonic acid as described in "Materialsand Methods." The cells were washed and incubated for 1 or 2 hr in the presenceof DMSO or TPA (10~7 M), and phospholipids were isolated and separated asdescribed in "Materials and Methods."

Radioactivity (dpm/dish x 10"3)

DMSO (1 hr) TPA (1 hr) DMSO (2 hr) TPA (2 hr)

CompoundPC

Lyso-PC

PELyso-PE

PIPSPA»H82.1a

10.412.6

6.50.54.71.8"C67.5

2.317.50.92.75.42.33H66.9

13.315.610.50.62.62.1"C57.0

2.726.1

1.13.04.32.53H62.8

12.913.09.30.66.52.214C60.3

2.720.0

1.02.98.02.63H52.6

15.931.712.60.74.02.414C48.3

3.129.6

1.14.36.43.2

a Mean of duplicate determinations.

An unexpected finding was the marked stimulated by TPA of32P,incorporation into both PE and lyso-PE. This stimulation wasnot accompanied by increased incorporation of [3H]ethanolamineor [3H]serine. These 2 precursors feed into the pathways whichwould be expected to lead to labeling by 32P,.One explanation

of this data is the existence of a base exchange reaction betweenPC and ethanolamine (13), and our data provide some supportfor this possibility. However, one aspect of the results requirescomment. As can be seen from the data in Table 1, TPA causesan extensive accumulation of 32P,label in PE and lyso-PE. In fact,

at all time points examined, radioactivity associated with these2 compounds is considerably greater than that associated withPC. This is not consistent with a simple base exchange reactionbetween ethanolamine and a uniformly 32Prlabeled PC pool,

because the absolute level of PC in HeLa cells exceeds that ofPE, and neither level is altered by TPA (unpublished data).Consequently, if labeling of PE is solely a consequence of baseexchange, it must involve a subpool of PC which is extensivelylabeled with 32P,.

The use of the PC-specific exchange protein to label the outer

monolayer of intact cells has considerable promise. Most studieson phospholipid metabolism involve prelabeling cells with 32P¡or

with labeled fatty acid or nitrogenous head groups. Any changesinduced by test substances must, therefore, be observed againstan extensive background labeling of the phospholipids. It is asubstantial advantage to be able to insert a phospholipid, labeled

in a defined way, into the external monolayer of the plasmamembrane and then to follow its redistribution into other phospholipids on stimulation of the cells. There may also be somelimitations associated with studies using cells labeled with exogenous PC. Presumably, such labeling will be dependent onlyon accessibility to the exchange protein, and may, therefore,"hide" the possible existence of metabolically distinct PC pools.

It is clear that TPA causes major changes in phospholipidmetabolism in culture cells. In addition to base exchange, TPAstimulates the turnover of PC initiated by phospholipase C (7, 8,15) and the breakdown of PC by phospholipase A2 (2, 6). Thesechanges might be expected to modify the activities of intrinsicmembrane proteins and to alter the interactions of membraneswith cytoplasmic proteins.

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1983;43:5564-5569. Cancer Res   Graeme R. Guy and Andrew W. Murray  HeLa CellsEffects of a Tumor Promoter on Phospholipid Metabolism in

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