Phospholipid analogues: side hain and polar head g ro u pd e pe n d e n t effets on phosphatidylholine biosynthesis Christoph C. Geilen,'. * Anne Haase,* Thomas Wieder,* Dietrih Arndt,t Reiner Zeisig,t and Werner Reutter* Institut fur Molekularbiologie und Biohemie der Freien Universitat Berlin,* D14195 BerlinDahlem, Germany; and Max Delbriik Centrum fur Molekulare Medizin,f BerlinBuh, Germany Abstrat In reent studies we showed that the phospholipid analogue hexadeylphosphoholine inhibits phosphatidylholine biosynthesis by affeting the transloation of the ratelimiting enzyme of phosphatidylholine biosynthesis, CTP:phosphoholine ytidylyltransferase (EC 2.7.7.15), to membranes, where it is ative (Geilen et al. 1992. J. Biol. Cha. 267: 67196724). The present study was performed to investigate the struturedependeny of this effet. It is shown that the inhibitory properties of phospholipid analogues are dependent on their alkyl side hain length (dodeylphosphoholine < tetradeylphosphoholine < hexadeylphosphoholine < heptadeylphosphoholine < otadeylphosphoholine > eiosadeylphosphoholine). Furthermore, it is demonstrated that this inhibition of phosphatidylholine biosynthesis by phospholipid analogues is also dependent on the polar head group (hexadeylphosphoholine > > hexadeylphosphoethanolamine = hexadeylphosphaserine). These effets result from an inhibition of the CTP:phosphoholine ytidylyltransferase and are not due to an inhibition of holine uptake or differenes in the ellular uptake of the phospholipid analogues investigated.geilen, C. C., A. Haase, T. Wieder, D. Arndt, R. Zeisig, and W. Reutter. Phospholipid analogues: side hain and polar head groupdependent effets on phosphatidylholine biosynthesis. J Lipid Res. 1994. 35: 625632. Supplementary key words ytotoxiity hexadeylphosphoholine ell proliferation CTP:phosphoholine ytidylyltransferase (EC 2.7.7.15) (CT) is the ratelimiting enzyme in the biosynthesis of phosphatidylholine in higher eukaryotes (for review see refs. 13). Its ativity is regulated by transloation of the enzyme between ytosol and membranes. The ytosoli, lipidfree form is inative, whereas the membranebound form is ative. Reently, a third form was desribed, whih is an ative, ytosoli enzyme112 kda protein aggregate (4). The transloation proess has been shown to be regulated by various lipids (5, 6). The lipids that were first reported to regulate CT were glyerophospholipids (79). Transloation to membranes is enhaned in partiular by the presene in the membrane of anioni glyerophospholipids suh as phosphatidylglyerol, phosphatidylserine, or phosphatidylinositol. Free fatty aids have also been reported to regulate CT ativity (112), and their ativating properties are ayl hain lengthdependent (6). Furthermore, mono and diaylglyerols may ativate CT (13, 14). It has been suggested that an inreased diaylglyerol ontent of ellular membranes after treatment of ells with phospholipase C (15) or phorbol ester (16) is the underlying mehanism of enhaned phosphatidylholine biosynthesis in these ells. These data on the ativation of CT by naturally ourring lipids in vivo and in vitro suggest that the proess of CTmembrane interation depends on both the surfae harge of membranes and the lipid paking of these membranes. Reently, the syntheti phospholipid, hexadeylphosphoholine (HePC), has been shown to inhibit PC biosynthesis (17, 18). This lipid analogue has also been reported to inhibit ell proliferation and tumor growth (1921). In the present study, we systematially investigated the effet of this lass of phospholipid analogues on phosphatidylholine biosynthesis and ell proliferation in vivo by using analogues that differ from hexadeylphosphoholine in their alkyl hain length or in their polar head group (Fig. l). Abbreviations: CT, CTP:phosphoholine ytidylyltransferase; HPLC, high performane liquid hromatography; HePC, hexadeylphosphoholine; PC, phosphatidylholine; HepPC, heptadeylphosphoholine; OPC, otadeylphosphoholine; EiPC, eiosadeylphosphoholine; DoPC, ddeylphosphoholine; TePC, tetradeylphosphoholine; HePS, hexadeylphosphoserine; HePE, hexadeylphosphoethanolamine; CMC, ritial miellar onentration; HFTLC, high performane thinlayer hromatography; TPA, 12tetradeanoylphorbol13aetate; PBS, phosphatebuffered saline. 'To whom orrespondene should be addressed at: Department of Dermatology, University Medial Center Steglitz, The Free University of Berlin, Hindenburgdamm 3, D122 Berlin, Germany. Journal of Lipid Researh Volume 35, 1994 625
Alkylphosphoholines: n = 1 DoPC n = 3 TePC n = 5 HePC n = 6 HepPC n = 7 OPC n = 9 EiPC Hexadeylphosphoethanolamine Hexadeylphosphoserine Fig. 1. Materials Chemial strutures of different phospholipid analogues. MATERIALS AND METHODS Hexadeylphosphoholine (HePC), heptadeylphosphoholine (HepPC), otadeylphosphoholine (OPC), and eiosadeylphosphoholine (EiPC) were synthesized as desribed reently (22, 23). Dodeylphosphoholine (DoPC) was prepared by Prof. J. Mulzer and M. Mikeleit, Institut fur Organishe Chemie, Freie Universitat Berlin. Tetradeylphosphoholine (TePC) was obtained from Novabiohem (Bad Soden, Germany). Hexadeylphosphoserine (HePS) and hexadeylphosphoethanolamine (HePE) were a gift from Asta Pharma (Frankfurt a. Main, Germany). The purity of eah analogue was heked by silia gelhplc onneted with a lightsattering detetor Sedex 55 (ERC, Regensburg, Germany) using nhexaneisopropanol 3:2 (v/v) as eluent. The detetion sensitivity was < 1 pmol of the analogue. All analogues investigated had a purity of more than 95%. Additionally, the identity of eah analogue was heked by mass spetrometry and 'HNMR analysis. [Methyl 3Hlholine hloride (2.83.1 TBq/mmol) was from Amersham (Braunshweig, Germany). Silia gel 6 HPTLC plates, solvents, and reagents (reagent grade) were purhased from E. Merk (Darmstadt, Germany) and Fluka (NeuUlm, Germany). The biinhonini aid (BCA)kit for protein determination was obtained from Piere (Weiskirhen, Germany). Misellaneous lipids and phos phatidylholine preursors were from Sigma (Munhen, Germany). For quantifiation of radioativity, a Berthold LB 2821 HR thinlayer hromatography sanner (Berthold; Wildbad, Germany) was used. Determination of the ritial miellar onentration (CMC) The ritial miellar onentration of alkylphosphoholines with different hain lengths was determined by the method of Saski and Shah (24). Briefly, eah sample was dissolved in aqueous, saturated iodine solution in a onentration range of.5% to.1% (weight per volume). The light transmission of the samples at 36 nm was ompared with that of iodine solution without additions. The onentrations were plotted halflogarithmially versus transmission. The intersetion of the two straight lines indiated the CMC value of the respetive substane. Cell ulture MDCK ells were grown in plasti ulture dishes (Nun, Wiesbaden, Germany) ontaining Dulbeo's minimal essential medium supplemented with 1% alf serum,.56 g/l Lglutamine, 1, IU peniillin, and.1 g/l streptomyin. Media and ulture reagents were obtained from Gibo (Berlin, Germany); peniillin and streptomyin were from Boehringer (Mannheim, Germany). Confluent ells were subultured every 3 days after detahing the ells with a trypsin/edta solution. For experimental purposes, ells were used on the third day of ulture. All phospholipid analogues were dissolved in PBS, ph 7.4, and then added to the ulture medium. Cytotoxiity assay Cytotoxiity of the different phospholipid analogues to MDCK ells was estimated aording to Culvenor et al. (25) by measurement of the alkaline phosphatase ativity of the ells. After inubation for 24 h with different onentrations of the respetive phospholipid analogue, the ells were entrifuged at 1 g for 1 min, and the supernatant was disarded. The reation mixture ontaining 2% diethanolamine, 2 pmol/l MgC12, 2 pmol/l p nitrophenolphosphate, and 1% Triton X1 was added. After 1 h at 37OC the reation was stopped with.5 mol/l NaOH. The amount of pnitrophenolate was determined photometrially at 41 nm. Cell proliferation assay Cell proliferation was measured by determination of the ell number aording to the method of Gilles, Didier, and Denton (26). Cells were fixed by treatment with 1% glutaraldehyde for 15 min, stained for 3 min in.1% rystal violet solution, then washed with deionized water for 15 min. Crystal violet absorbed by ell nulei was solubilized with.2% Triton X1 and the amount released was quantitated photometrially at 59 nm. 626 Journal of Lipid Researh Volume 35, 1994
Choline uptake assay MDCK ells were grown to onfluene on ollagenoated plasti foils. For the assay, ells were treated with medium ontaining 5 pmol/l of the respetive lipid analogue for 2 min and then were immersed in pulse medium ontaining 28 pmol/l holine and 3 pci/ml [3Hmethyl] holine as radioative traer. After different times, as indiated in the legends, ells were washed thoroughly twie with 58 pmol/l holine in PBS. For quantifiation of the holine uptake, ells with the entire plasti foils were taken for sintillation ounting. Uptake was alulated by the inorporated traer radioativity. Measurement of ellular uptake of the different phospholipid analogues MDCK ells were grown to onfluene and treated with medium ontaining 5 pmol/l of the respetive lipid analogue for 6 h. Then, ells were washed twie with PBS, harvested with a ell lifter (Costar; Cambridge, MA), and lyophilized. The pellets were extrated by modified lipid extration aording to Bligh and Dyer (27): 5 p1 methanol, 25 p1 hloroform, and 2 p1 water were added. Samples were stirred for 2 min on a vortexmixer and entrifuged at 13, g for 1 min. Phase separation was aomplished by addition of 25 pl hloroform and 25 pl water. The suspension and entrifugation steps were repeated. Then 15 pl of both the organi and the aqueous phases were applied to silia gel 6 HPTLC plates using an HPTLC appliator (Linomat 111; Camag, Berlin, Germany). Lipids were separated aording to Touhstone, Chen, and Beaver (28), using the solvent hloroformmethanoltriethylaminewater 3:35:34:8 (by vol). Phospholipid analogues were stained with upri sulfate in phosphori aid and quantified by use of a videodensitometer (BioteFisher, Reiskirhen, Germany). Staining was linear in the range of.2 to 2. nmol of eah analogue. Measurement of phosphatidylholine biosynthesis After inubation with pulsemedium ([ 3H]holine; 1 pci/ml) supplemented with 5 pmol/l of the different phospholipid analogues, ells were washed twie with ieold PBS (ph 7.2) and harvested with a ell lifter (Costar; Cambridge, MA) followed by modified lipid extration aording to Bligh and Dyer (27) as desribed above. Then 1 pl of the hloroform phase was taken for sintillation ounting and 15 pl was applied to silia gel 6 HPTLC plates using an HPTLC appliator (Linomat 111; Camag, Berlin, Germany). Lipids were separated aording to Touhstone et al. (28) as desribed above. Radioativity was quantified by radiosanning (LB 2821 HR, Berthold, Wildbad, Germany). Phospholipids were identified by alibrating the sanner with known standards. Approximately 95 % of the radioativity ohromatographed with the phosphatidylholine standard. For determining PC biosynthesis under stimulating onditions, ells were simultaneously treated with 5 nmol/l 12Otetradeanoylphorbol13aetate (TPA) and 5 pmol/l of the different alkylphosphoholines. Permeabilization of ells by digitonin Confluent MDCK ells were inubated for 6 h in medium ontaining no supplement (ontrol) or 5 pmol/l of the respetive phospholipid analogue. Cells were then washed two times with ieold PBS, and all the following steps were arried out at 4OC. Three hundred pl buffer ontaining 5 mm TrisHC1 (ph 7.4), 15 mm NaCl, 1 mm EDTA, 2 mm dithiothreitol,.25% NaN3, 1 mm PMSF, and.5 mg/ml digitonin was added to the ells. After 3 min the digitonin supernatant was removed. Undissolved digitonin was pelleted at 13, g for 2 min and the remaining ell ghosts were washed with 1 ml of PBS, harvested in 5 pl of buffer as desribed above (the harvesting buffer ontained.3% Triton X1 and no digitonin) and then homogenized by a Doune homogenizer. C ytidyltransferase ativity was measured in the digitonin supernatants and in the ell ghost homogenates as desribed below. From the data, the ratio of ytosoli and membranebound CT ativity was alulated for eah dish. Cytidylyltransferase assay Cytidylyltransferase ativity was measured by a modified method of Sohal and Cornel1 (29). The reation mixture ontained 5 mmol/l TrisHC1, ph 7.4,.3% Triton X1, 1 mmol/l NaCl, 1 mmol/l MgClP, 3 mmol/l CTP, 1.5 mmol/l [methyl14c]phosphoholine (sp at 2 Bq/nmol), liposomes (4 pmolll phosphatidylholine4 pmolll olei aid) and 1 11 of enzyme preparation in a final volume of 55 p1. After inubating for 3 min at 37OC, the reation was stopped by freezing the samples in liquid nitrogen, The samples were lyophilized, dissolved in 2 pl methanolwater 1:l (v/v) and applied to HPTLC plates. After developing the plates in a solvent system ontaining methanol.6% NaC125% aqueous NH3 8:5:1 (by vol), the radioativity was determined by radiosanning. One unit of enzyme ativity is defined as one nmol of CDPholine formed per min. Other proedures Cellular protein was determined by the BCAassay (3) using bovine serum albumin as a standard. Statistial analysis was performed by the Student's ttest. Geilen et al. Phospholipid analogues and phosphatidylholine biosynthesis 627
I"" I 8 6 4 2 1 8 6 4 9 1 n (I) Fig. 2. Antiproliferative effet of different phospholipid analogues. MDCK ells were inubated with 1 pmolll of the respetive phospholipid analogue. For ontrol experiments no supplements were added. After 16 h of inubation the proliferation rate was determined by rystal violet assay as desribed in Materials and Methods. Values are given as 76 inhibition of ell proliferation ompared with ontrol values + SD (n = 4). RESULTS Dosedependent effet of different phospholipid analogues on ell proliferation and ell toxiity The dosedependent effet of phospholipid analogues with different hain length and various polar head groups on the proliferation of MDCK ells was investigated. Conentrations of 1 pmol/l, 5 pmol/l, 1 pmol/l, and 2 pmolll were used and the ells were inubated for 16 h. All phospholipid analogues investigated inhibited the proliferation of MDCK ells in a dosedependent manner, but the inhibitory ativity of the different phospholipid analogues varied over a wide range. At a onentration of 1 pmolll, the phospholipid analogues with a serine and ethanolamine head group, HePS and HePE, showed an inhibition rate of less than 2% versus ontrols (IC5 > 2 pmol/l) (Fig. 2). The effet of the holine derivatives was dependent on the length of the alkyl group (Fig. 2). Short phospholipid analogues suh as DoPC and TePC were weak inhibitors of ell proliferation with IC5 values above 2 pmol/l. Potent substanes were HePC, HepPC, and OPC, whih showed IC5,, values of about 18 pmolll, 1 pmol/l, and 75 pmol/l, respetively. A further inrease in hain length resulted in a derease of the antiproliferative effet. The IC5 of EiPC was above 2 pmol/l. To examine the ytotoxi effet of the different phospholipid analogues, onfluent MDCK ells were inubated with phospholipid analogue onentrations of 1 pmol/l3 pmolll for 6 h. Subsequently, viability of the ells was determined. A dosedependent inrease of the ytotoxi effet ourred for all substanes examined. With exeption of HepPC, no signifiant ytotoxi effets ourred at onentrations 5 5 pmolll (viability 2 9%) for all lipid analogues tested. In general, the results were similar to those of the ell proliferation assay. However, at 5 pmol/l the most ytotoxi agent was HepPC (Fig. 3). 12 11, P 11 1 9 8 7 6 5 4 TePC 2 3El. 1 A EPC,.,.,, o l 9 2 4 6 8 1 12 2 4 6 8 1 12 Conentration [~"lll] Conentration [pmol/l] Fig. 3. Cytotoxi effet of different phospholipid analogues. MDCK ells were inubated for 6 h with different onentrations of the respetive phospholipid analogues as indiated in the figure (DoPC; TePC; HePC; HepPC; OPC; EiPC; HePS; HePE). Alkaline phosphatase ativity was then determined as desribed in Materials and Methods. Values are the mean of four separate determinations (SD < 5%). 8 6 628 Journal of Lipid Researh Volume 35, 1994
a. 2 2 o Q 2 3 a)= 4 ". 9 U. I 44 P g 1 "= 1 "Z J =, 8 u + 5 ", 4 a a )o ([Io 3 U p I& 9. C._ 4 4 u z a a a?'a,. 8 4;.p $ 6 I 4._ * I 1 P 6 P 2 8 4._ I 2 48 2 n A Fig. 4. A: Effet of different phospholipid analogues on holine inorporation into PC under phorbol esterstimulated onditions. For the experiments under TPAstimulated onditions, ells were pretreated with 5 nmol/l TPA and 5 pmoln o f the respetive phospholipid analogues simultaneously for 3 h. Then radiolabeled holine (2 pcilml) was added and the ells were inubated further for 2 h. T h e subsequent steps were arried out as desribed for the experiments under nonstimulated onditions (Fig. 4B). T h e values of inorporated radioativity are given in dpmlpg protein ~tsd (n = 3). (Signifiantly different from TPAstimulated ontrol: *P 5.5.) B: Effet of different phospholipid analogues on holine inorporation into PC under nonstimulated onditions. M D C K ells were inubated for 6 h with 5 pmol/l of the respetive phospholipid analogue and [methylshiholine (2 pcilml). T h e ells were harvested, lipids were extrated, and the holine inorporation into phosphatidylholine was determined as desribed in Materials and Methods. For ontrol values no phospholipid analogue was added. T h e values of inorporated radioativity are given in % of ontrol and are the mean of three separate determinations SD. (Signifiantly different from ontrol: *P 5.5; **P5.1.) * Effet of alkylphosphoholines with different alkyl side hains on TPAstimulated and nonstimulated phosphatidylholine biosynthesis MDCK ells were pretreated for 3.h with 5 nmol/l TPA, or with 5 nmol/l TPA plus 5 pmol/l of the respetive phospholipid analogue, or with no supplements as ontrol. Then [methyl3h]holine [2.5 pci/ml] was added. After 2 h of inubation, ells were harvested and PC was analyzed as desribed in Materials and Methods. The results shown in Fig. 4A indiate that antagonization of TPAstimulated PC biosynthesis is dependent on the length of the alkyl side hain, with a maximum effet at C18 (OPC). Nearly the same results were obtained with nonstimulated MDCK ells. After 6 h of inubation with different phospholipid analogues, inorporation of [ methyl3hlholine into PC was inhibited. The inhibition depended on hain length (Fig. 4B), and OPC was the most effiient inhibitor (7% inhibition). Beside the fat that DoPC has no effet on nonstimulated PC biosynthesis, it abolished TPAstimulated PC biosynthesis by approximately 6%. All phospholipid analogues investigated had no effet on holine uptake (data not shown). Effet of phospholipid analogues with different polar head groups on phosphatidylholine biosynthesis To determine the effet of different polar head groups on holine inorporation, HePC, HePS, and HePE were Geilen et al. investigated. The experiments were arried out as desribed in the previous setion. The results shown in Fig. 4B indiate that the holine head group is essential for the inhibitory effet on PC biosynthesis. This is also true for the antagonization of phorbol ester stimulation (data not shown). 3 4 35 m Fig. 5. Effet of different alkylphosphoholines on the subellular distribution of the CTP:phosphoholine ytidylyltransferase. MDCK ells were inubated for 6 h with 5 pmolll of the respetive phospholipid analogue. Then the subellular distribution of C T was determined by digitonin release as desribed in Materials and Methods. The values of membranebound enzyme are given in % of the total ativity of C T f SD (n = 3). (Signifiantly different from ontrol: *P 5.5.) Phospholipid analopes and phosphatidylholine biosynthesis 629
Effet of alkylphosphoholines on the subellular distribution of CTP:phosphoholine ytid yly ltransferase By permeabilizing MDCK ells with digitonin, whih auses ytosoli ontents to leak into the ulture medium, it is possible to distinguish between the ytosoli and the membranebound form of CT. In order to examine the effet of different alkylphosphoholines on the transloation proess, the distribution of CT in ontrol and alkylphosphoholinetreated ells (6 h) was measured by determination of CT ativity in the digitonin supernatant and in the ell ghosts. Fig. 5 shows the side haindependent effet of different alkylphosphoholines on the distribution of CT ativity after a digitonin release of 3 min, whih is in aordane with the results of the inhibition of PC biosynthesis desribed above. Cellular uptake of different phospholipid analogues To exlude the possibility that differenes in the ation of phospholipid analogues investigated in this study were due to differenes in their ellular uptake, this ellular uptake was determined as desribed in Materials and Methods. As shown in Table 1, all phospholipid analogues, with the exeption of DoPC, were taken up to a similar extent. There was no signifiant orrelation between uptake of phospholipid analogues and inhibition of ell proliferation (r =.41), and between uptake of phospholipid analogues and inhibition of PC biosynthesis (r =.7). DISCUSSION In this study, we investigated the effet of different phospholipid analogues on phosphatidylholine biosynthesis and ell proliferation in vivo. Previous studies showed that the syntheti phospholipid analogue, hexadeylphosphoholine, disturbs the transloation proess of the key enzyme of PC biosynthesis, CTP:phosphoholine ytidylyl TABLE 1. Phospholipid Analogue DoPC TePC HePC HepPC OCPC EiPC HePE HePS Cellular uptake of different phospholipid analogues Uptake nmol analogue/1" ells 2.1 C.1 7.3 2.8 7.3.7 6.4 C.5 11. +_.8 16.2 1.2 1.4 +_ 1.8 5.2?.6 Confluent MDCK ells were treated with medium ontaining 5 pmol/l of the respetive phospholipid analogue for 6 h. Cells were harvested, extrated, and ellular uptake was measured by HPTLC as desribed in Materials and Methods (n = 3). by about 6%. transferase, and finally leads to an inhibition of PC biosynthesis in vivo (18). Furthermore, different studies have shown that HePC inhibits the ell proliferation of various ell lines (1921). CT is an amphipathi protein and its ativation is lipiddependent. In most reported studies on lipid requirements, naturally ourring lipids suh as glyerophospholipids, fatty aids, or diaylglyerols were used, and the results of these studies have led to several onlusions about CTmembrane interations (5, 6). In the present study, we tested these onlusions using syntheti phospholipid analogues. By omparing our data with published results, the following points were onfirmed. First, it was reported that lipids that at as transloators have an optimum hain length of > 14 arbon atoms. For example, olei aid has the strongest stimulatory effet on PC biosynthesis (9). In our results,opc showed the best inhibitory effet on holine inorporation into PC as well as on the CT distribution, where OPC redued the membranebound form by over 5% ompared to ontrol ells. It is possible that a lipid that modulates CT ativity must have an optimum hain length to be integrated into the membrane bilayer and its effet on the regulation of CT transloation depends on its polar head group. Choline head groupontaining lipids, suh as alkylphosphoholines, alkyllysophosphoholines, or PC, inhibit CT transloation to membranes, whereas only lipids without a holine headgroup, suh as olei aid, oleoyl alohol, or diaylglyerol, ould ativate the membrane transloation of CT. Very reently, Jamil, Hath, and Vane (31) suggested that the binding of CT to membranes is modulated by the ratio of bilayerforming lipids (e.g., PC) to nonbilayerforming lipids (e.g., olei aid or diaylglyerol). The phospholipid analogues investigated are nonbilayerforming lipids (32), but differed in their ation on CT transloation and PC biosynthesis. From our data, we therefore suggest that the ratio of holine head groupontaining lipids to nonholine head groupontaining lipids modulates CT transloation. However, an effet on holine uptake ould be exluded, whih is in aordane with our previous findings in studies with HePC (18). Furthermore, no differenes ourred in the ellular uptake of the phospholipid analogues that ould explain their differenes in ation. In a reent study, we showed that HePC antagonizes phorbol esterstimulated PC biosynthesis by inhibiting the transloation of CT (33). Therefore, we investigated the effet of the different alkylphosphoholines in TPAstimulated MDCK ells. The hain lengthdependent effet was similar to that found in experiments with nonstimulated ells. However, it is obvious that the response to DoPC is different in stimulated and nonstimulated ells, D~PC has no effet On p biosynthesis in nonbut it antagonizes the TPAindued effet 63 Journal of Lipid Researh Volume 35, 1994
Furthermore, the observation that lipids with the same polar head group suh as HePC, OPC, and lpalmitoyllysophosphatidylholine, have different effets on CT transloation has to be investigated in more detail. It was suggested that hanges in membrane properties, like fluidity and paking of lipids, influene the type of regulative ation; but HePC, OPC, and LPC have nearly the same biophysial properties. For example, all alkylphosphoholines investigated have ritial miellar onentrations in the range between 17 and 28 kmol/l measured by the method of Saski and Shah (24). Our results on the effet of this lass of phospholipid analogues with different polar head groups support the suggestion that the struture of the polar head group (e.g., harge, spae requirements) of the lipid influenes its modulatory properties. HePS and HePE do not inhibit PC biosynthesis in TPAstimulated and nonstimulated MDCK ells. Conerning CTmembrane interation, it is interesting that CT ativity depends on the state of the lipid phase. It was shown in in vitro studies that fluidphase lipids are better ativators of CT than gelphase lipids. We demonstrated that more than 95% of the inorporated HePC is assoiated with ellular membranes (34). Therefore, we suggest that phospholipid analogues suh as HePC are inorporated into ellular membranes and inhibit the CTmembrane fluidity. At least, we demonstrated that the antiproliferative effet of alkylphosphoholines is paralleled by their ability to inhibit PC biosynthesis. There is a good orrelation between the effet of lipid analogues on ell proliferation and inorporation of radiolabeled holine into PC (7 =.96) and between the effet on CT transloation and inorporation of radiolabeled holine into PC (7 =.92). In ontrast, no signifiant orrelation ourred between the effet on ell viability and inorporation of radiolabeled holine into PC (r =.69), and between ell viability and ell proliferation (7 =.6). In previous studies, it was suggested that the antiproliferative effet of HePC is due to an inhibition of protein kinase C (35, 36). From our data, we suggest that the antiproliferative effet of alkylphosphoholines might also be mediated by inhibition of PC biosynthesis. In further studies, we will fous our interest on the relationship between PC biosynthesis and ell proliferation. Blll This work was supported by a grant of the Deutshe Forshungsgemeinshaft (Ge 641/31). We thank Dr. T. A. Sott for improving the English style of the manusript. Manusript reeived 21 June 1993 and in revised form I1 Otobn 1993. REFERENCES 1. Vane, D. E. 199. Phosphatidylholine metabolism: masohisti enzymology, metaboli regulation, and lipoprotein assembly. Biohem. Cell Biol. 68: 11511165. 2. Kent, C. 199. 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