Báo cáo khoa học: Hexadecylphosphocholine inhibits phosphatidylcholine biosynthesis and the proliferation of HepG2 cells pot

Hexadecylphosphocholine inhibits phosphatidylcholine biosynthesis and the proliferation of HepG2 cells Jose ´ M. Jime ´ nez-Lo ´ pez, Marı ´ a P. Carrasco, Josefa L. Segovia and Carmen Marco Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Granada, Spain Hexadecylphosphocholine (HePC) is a synthetic lipid rep- resentative of a new group of antiproliferative agents, alkylphosphocholines (APC), which are promising candi- dates in anticancer therapy. Thus we have studied the action of HePC on the human hepatoblastoma cell line HepG2, which is frequently used as a model for studies into hepatic lipid metabolism. Non-toxic, micromolar concentrations of HePC exerted an antiproliferative effect on this hepatoma cell line. The incorporation into phosphatidylcholine (PC) of the exogenous precursor [methyl- 14 C]choline was substan- tially reduced by HePC. This effect was not due to any alteration in choline uptake by the cells, the degradation rate of PC or the release of PC into the culture medium. As an accumulation of soluble choline derivatives points to CTP:phosphocholine cytidylyltransferase (CT) as the target of HePC activity we examined its effects on the different enzymes involved in the biosynthesis of PC via CDP–cho- line. Treatment with HePC altered neither the activity of choline kinase (CK) nor that of diacylglycerol cholinephos- photransferase (CPT), but it did inhibit CT activity in HepG2 cells. In vitro HePC also inhibited the activity of cytosolic but not membrane-bound CT. Taken together our results suggest that HePC interferes specifically with the biosynthesis of PC in HepG2 cells by depressing CT trans- location to the membrane, which may well impair their proliferation. Keywords: alkylphosphocholines; CTP:phosphocholine cytidylyltransferase; phosphatidylcholine metabolism; human hepatoblastoma cells; proliferation. Hexadecylphosphocholine(HePC)isanewmembrane-active antineoplastic compound belonging to the alkylphospho- cholines (APC) group, which exert antitumoral activity against a broad spectrum of established tumour cell lines [1]. It is currently used for the topical palliative treatment of cutaneous metastases of mammary carcinomas [2]. There is growing interest in the biological activity of these lipid analogues as they do not interact with DNA but selectively inhibit the growth of transformed cells and could well complement existing DNA-directed anticancer chemo- therapies. APC administered either orally or intravenously accu- mulate in different organs, including the liver [3]. Although at present the systemic application of HePC to cancer patients is limited because of the significant gastrointestinal intolerance it often entails, alternative formulations of APC treatment towards a variety of tumours are currently being developed [4,5]. A wide variety of cytotoxic mechanisms have been attributed to this class of antineoplastic compounds [6]. Their direct incorporation into the cell membrane, which seems to be the primary site of their activity, suggests that the molecular mechanism behind their effect involves some membrane-dependent function. There is considerable evi- dence to suggest that HePC could interfere with an early step in lipid-signal transduction events, which might therefore be responsible for its capacity to inhibit growth [7]. Previous reports have indicated the ability of HePC to interfere with phospholipid metabolism. However, a clear mechanism of action has not been established yet. An important mechanism for HePC-induced biological effects may be the inhibition of phospholipase C [8], phospholipase A 2 [9] or the activation of phospholipase D [10,11] which may be achieved by protein kinase C (PKC) dependent or independent mechanism, depending of the cell line investi- gated [11]. The toxicity of HePC may also be related to a disruption of calcium homeostasis [12]. Other authors have shown that exposure of cells to HePC leads to a reduction in the biosynthesis of phosphatidylcholine (PC) in MDCK [13], HaCaT [14] and HL60 cells [15] by inhibiting the rate- limiting enzyme CTP:phosphocholine cytidylyltransferase (CT). In neuronal axons, on the other hand, HePC does not seem to act in the same way but rather blocks choline uptake by the cell [16]. In the light of these findings, our previous experience with hepatic cells and research into the action of different xenobiotics on phospholipid metabolism [17,18] has promp- ted us to investigate the effect of HePC on the tumoral Correspondence to C. Marco, Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Granada, Av. Fuentenueva s/n, Granada 18071, Spain. Fax: + 34 958249945, Tel.: + 34 958243086, E-mail: cmarco@ugr.es Abbreviations: APC, alkylphosphocholines; CK, choline kinase; CP, choline phosphate; CT, CTP:phosphocholine cytidylyltransferase; CPT, diacylglycerol cholinephosphotransferase; EMEM, Eagle’s minimum essential medium; HePC, hexadecylphosphocholine; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; SM, sphingomyelin. Enzymes: choline kinase (CK) (ATP:choline phosphotransferase, EC 2.7.1.32); CTP:phosphocholine cytidylyltransferase (CT) (EC 2.7.7.15); diacylglycerol cholinephosphotransferase (CPT) (CDP–choline:1,2-diacylglycerol cholinephosphotransferase, EC 2.7.8.2) (Received 15 May 2002, revised 18 July 2002, accepted 2 August 2002) Eur. J. Biochem. 269, 4649–4655 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03169.x hepatic cell line HepG2. We studied its cytotoxic and cytostatic activity against HepG2 cells and examined its influence on de novo PC biosynthesis by using the precursor [methyl- 14 C]choline as radioactive marker. We measured the effects of HePC on choline incorporation into soluble intermediates in the CDP–choline pathway as well as PC, the final product of this biosynthetic pathway. We also analysed the effect of HePC on the enzyme activities involved in the de novo biosynthesis of PC. MATERIALS AND METHODS Materials [methyl- 14 C]choline chloride (55 CiÆmol )1 ), [methyl- 14 C] choline phosphate (56 CiÆmol )1 ) and CDP-[methyl- 14 C]cho- line (54 CiÆmol )1 ) were supplied by Amersham Pharmacia Biotech (Freiburg, Germany). Fetal bovine serum was from Roche Diagnostics (Barcelona, Spain). HePC, Eagle’s minimum essential medium (EMEM) and thin-layer chromatography (TLC) plates were from Sigma-Aldrich (Madrid, Spain). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide (MTT) was from Molecular Probes (Leiden, The Netherlands). All other reagents were of analytical grade. Cell culture The human hepatoma cell line HepG2 was obtained from the European Collection of Animal Cell Cultures (Salisbury, Wiltshire, UK). Cells were routinely grown in EMEM supplemented with 10% v/v heat-inactivated fetal bovine serum, 2 m ML -glutamine, 1% nonessential amino acids, and an antibiotic solution (100 unitsÆml )1 penicillin and 100 lgÆmL )1 streptomycin) at pH 7.4 (complete medium). Cells were seeded on tissue-culture plates (Nunc TM )for adherent cells at densities of 3 · 10 4 cellsÆcm )2 and incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 . The medium was replaced every 2 days and the cells were subcultured by trypsinization before confluence. The cells were used in experiments after 6 or 7 days culture, by which time dense monolayers at about 70% confluence had formed (approximately 3 · 10 6 cells per 60-mm dish). HePC was dissolved in phosphate-buffered saline (NaCl/P i , pH 7.4) shortly before being added to the culture medium to the required final concentration. APC are readily bound to serum proteins such as albumin or lipoproteins, thus lowering their uptake rate into the cells and decreasing their biological activity. Therefore, all experiments were carried out with cells growing in medium supplemented with serum. Assays for cell viability and proliferation The cytotoxic effect of HePC on HepG2 cells was determined by the MTT test, based upon the conversion by viable cells of a tetrazolium salt into a blue formazan product [19]. The toxicity of HePC against HepG2 cells was also determined by the trypan blue exclusion assay and by measuring the release of lactate dehydrogenase (LDH) activity from the cytosol of damaged cells into the extra- cellular medium. Cell proliferation was determined by haemocytometer counting. [Methyl- 14 C]choline incorporation Cells were subcultured in 12-well culture plates and grown in EMEM/10% fetal bovine serum as indicated above. Cells in log-phase growth were incubated at 37 °Cfor6hin complete medium containing [methyl- 14 C]choline (60 l M , 55 CiÆmol )1 ) and supplemented with 50 or 100 l M HePC, whilst untreated cells were used as control. The medium was then withdrawn and the cells were washed twice and harvested by scraping with a rubber policeman into ice-cold NaCl/P i . Lipids were extracted from the cells following the procedure of Bligh and Dyer [20]. The chloroform layer was dried by evaporation with a stream of nitrogen whilst the water-methanol phase was collected and used to determine water-soluble metabolites. We analysed the incorporation of radioactive choline into PC and sphingomyelin (SM), and also into soluble intermediaries of the CDP–choline path- way. The different phospholipids were separated on silica- gel 60 G TLC plates using a mixture of chloroform/ methanol/acetic-acid/water (60 : 50 : 1 : 4, v/v) as solvent. The soluble metabolites from choline were separated by TLC developed in methanol/0.6% NaCl/25% aqueous NH 3 (50 : 50 : 5, v/v). The spots were made visible by exposure to iodine vapour and ultraviolet light, and assigned by means of standards. Radiometric measurements of lipid spots were made by liquid scintillation using a Beckman 6000-TA counter (Madrid, Spain). The values were normalized to the quantity of cell protein determined by Bradford’s method [21] with BSA as standard. Choline uptake assay Cells were preincubated for 5 min, 1.5 h or 6 h at 37 °C either in a medium containing 50 l M HePC or with no supplement as control. The medium was then removed and the cells immediately exposed to a medium containing [methyl- 14 C]choline (60 l M ,55CiÆmol )1 )for3minat 37 °C. The incorporation of [ 14 C]choline was stopped by medium aspiration followed by two washes with ice-cold NaCl/P i containing 580 l M choline. The lipids were extrac- ted directly from the attached cells according to Bligh and Dyer [20]. After lipid extraction the aqueous and organic phases were separated and the radioactivity of each phase was measured. Incorporation was determined as the total amount of radiolabel taken up by the cells. Enzyme activities in the Kennedy pathway Proliferating cells from 90-mm dishes were harvested by scraping into ice-cold NaCl/P i , collected by low-speed centrifugation and suspended in an ice-cold homogenizing buffer containing 0.145 M NaCl, 10 m M Tris/HCl, 1 m M EDTA, 10 m M KF, 0.2 m M phenylmethanesulfonyl fluoride (pH 7.4), by using a volume equal to four times the volume of the cellular pellet. Cells in suspension were sonicated for 2 s with a microprobe in an ice bath, and the homogenate was centrifuged immediately at 105 000 g for 30 min at 4 °C to yield a particulate fraction and the cytosolic supernatant. The membrane pellet was suspended in ice-cold 0.25 M sucrose, 10 m M Tris/HCl, 1 m M EDTA, 0.2 m M phenyl- methanesulfonyl fluoride (pH 7.4). The protein concentra- tion of each fraction was then measured. The determination of marker enzymes indicated that this centrifugation 4650 J. M. Jime ´ nez-Lo ´ pez et al.(Eur. J. Biochem. 269) Ó FEBS 2002 procedure resulted in less than 8% contamination of the microsomes and mitochondria in the cytosolic fraction. Choline kinase assay. Choline kinase (CK) activity was assayed by measuring the rate of [methyl- 14 C]choline incorporation into choline phosphate (CP) according to the method described by Pelech et al. [22], using approxi- mately 50 lg of cytosolic protein. CTP:phosphocholine cytidylyltransferase assay. CTP: phosphocholine cytidylyltransferase (CT) activity was determined in both the cytosolic and the particulate fraction by measuring the formation of radiolabelled CDP–choline from [methyl- 14 C]choline phosphate, as reported in Weinhold and Feldman [23], using approximately 25 lg of cytosolic or membrane protein. Enzyme activity in the cytosolic fraction was measured in the presence of PC/oleate liposomes (500 l M /500 l M ) in the final reaction mixture, unless stated otherwise, whilst membrane CT activity was assayed without liposomes. Diacylglycerol cholinephosphotransferase assay. Diacyl- glycerol cholinephosphotransferase (CPT) was assayed by measuring the rate of incorporation of CDP-[methyl- 14 C] choline into PC, according to the method reported by Cornell [24], using at least 50 lgofmembraneprotein. Data analysis Data are expressed as means ± SEM, as indicated in the figure and table legends. Statistical comparisons were made by variance analysis followed by the Bonferroni test using the SPSS 9.0 program. Values of P < 0.05 were considered to be statistically significant. RESULTS Effect of HePC on cell toxicity and cell proliferation We observed the toxic effect of 6 hours’ exposure to HePC in proliferating HepG2 cells by measuring the leakage of LDH into the extracellular medium and quantifying formazan production from MTT (Fig. 1). HePC concentrations of less than 100 l M caused no LDH to be released into the medium but at higher doses there was a concomitant increase in LDH activity. These results were confirmed by the formazan produced from MTT, which also indicated that the cells were affected by HePC at concentrations of more than 100 l M . Thus in subsequent experiments we used concentrations equal to or lower than 100 l M to avoid cell lysis. The antiproliferative effect of HePC has already been demonstrated in different tumour cells and cell lines [1] but data are still unavailable for tumoral hepatic cells. To examine the possible action of HePC on HepG2 prolifer- ation we treated the cells with 25, 50, or 75 l M concentra- tions for 48 h. A concentration of 25 l M did not change the growth rate but after 48 h concentrations of 50 and 75 l M had reduced cell numbers significantly (Fig. 2). This anti- proliferative action could not be put down to lysis as the decrease in the number of viable cells was not accompanied by a significant increase in nonviable cells, as measured by the trypan blue exclusion assay and LDH release into the medium. Inhibition of PC biosynthesis by HePC We examined the effect of HePC on the biosynthesis of PC by using choline as exogenous precursor. Cells were incuba- ted for 6 h with [methyl- 14 C]choline either in the presence or absence of HePC. HePC inhibited the incorporation of choline into both PC and SM in a dose-dependent manner (Fig. 3). As far as the soluble intermediates in the CDP– choline pathway are concerned, HePC caused a significant increase in the label of CP and a decrease in that of CDP– choline compared to controls (Fig. 3). These effects were markedly enhanced at a concentration of 100 l M HePC. It is interesting to note that the label in betaine, the product of choline oxidation, fell after exposure to 100 l M HePC (i.e. 0.77 ± 0.04 nmolÆmg protein )1 in control cells vs. Fig. 1. Cytotoxic effect of HePC on HepG2 cells. Cells growing in log- phase were incubated for 6 h with different concentrations of HePC. The release of LDH into the medium is given as absorbance at 340 nm (A) and formazan production from MTT as absorbance measured at 570 nm with background subtraction at 630 nm (B). Significant dif- ference from control is *P < 0.002. Fig. 2. Effect of HePC on the proliferation of HepG2 cells. Cells growing in log-phase were incubated with different concentrations of HePC. Total cell numbers were determined by counting in a haemo- cytometer. Results are expressed as means ± SEM for four inde- pendent samples in duplicate. Significant differences from controls are *P <0.01;**P < 0.001. Ó FEBS 2002 Hexadecylphosphocholine and HepG2 cells (Eur. J. Biochem. 269) 4651 0.64 ± 0.02 nmolÆmg protein )1 in 100 l M HePC-treated cells) whilst concentrations of 50 or 100 l M HePC failed to produce any alteration in the radioactivity associated to the choline pool (0.34 ± 0.03 nmolÆmg protein )1 in control cells). It has been widely demonstrated that oleate activates the PC synthesis in HepG2 cells increasing the CT activity. Thence, in another set of experiments we assayed the combined effect of HePC and oleate. As it can be observed in Table 1, exposure of HepG2 cells to oleate in the presence or absence of HePC, significantly increases the choline incorporation into PC. As oleate drastically reduces the inhibitory effect of the APC on the PC formation, the HePC 50 l M does not significantly alter the synthesis of this phospholipid in the presence of oleate. These results suggest that in HepG2 cells, HePC produces an alteration in PC biosynthesis via CDP–choline, although a modification in the degradation rate of newly synthesized PC could also contribute to this effect. To test this latter possibility we prelabelled the cells with [methyl- 14 C]choline for 24 h. The radioactive medium was removed and the labelled cells were incubated for an additional period of 6 h either in the presence or absence of HePC. The results indicate that the APC did not affect the rate of intracellular PC degradation as the radioactive label of PC was similar in both control and HePC-treated cells and neither was the secretion of labelled PC altered by exposure to HePC (results not shown). Effect of HePC on choline uptake by HepG2 cells To determine its possible effect on choline uptake the cells were exposed to HePC for different periods of time, pulsed with [methyl- 14 C]choline for 3 min and the radioactivity associated to the cells was measured. We chose this short time period to avoid intracellular choline metabolism. Exposure to 50 l M HePCforupto6hdidnotalter choline uptake by HepG2 cells to any significant extent. (i.e. 51.3 ± 2.6 pmolÆmin )1 per 10 6 cells in control vs. 50.3 ± 1.8 pmolÆmin )1 per 10 6 cells in HePC-treated cells). Effect of HePC on enzymes of the CDP–choline pathway According to the observations made above, and bearing in mind that the incorporation of choline into PC might be inhibited by HePC interfering with the biosynthesis of PC, we went on to analyse the enzyme activities involved in this synthetic pathway. Cells were exposed to 50 l M HePC for 6 h and the cytosolic and particulate fractions were obtained as described in the Materials and methods section. Neither CK assayed in the cytosol nor CPT activity measured in the particulate fraction were affected by HePC (Table 2). The exposure of HepG2 cells to HePC resulted in a significant inhibition in CT activity in the particulate fraction without affecting the activity of cytosolic CT. A good way of arriving at the real amount of soluble and membrane-bound CT in the cell is to calculate the total activity of both forms for each dish (3 · 10 6 cells). Thus we determined the effect of 50 l M and 100 l M doses of HePC upon the contribution of particulate and cytosolic CT to the Table 1. Reversion by oleate of effect caused by HePC on phosphati- dylcholine biosynthesis. Proliferating cells were incubated for 6 h in EMEM/10% fetal bovine serum containing [methyl- 14 C]choline (60 l M ,55CiÆmol )1 ) and different concentrations of HePC, in the presence or absence of oleate 1 m M BSA 1%. The incorporation of [ 14 C]choline into PtdCho was determined, as described in the Materials and methods section. Results are expressed as means ± SEM for four independent samples. nmol PCÆmg protein )1 None 50 l M HePC 100 l M HePC Without oleate 3.91 ± 0.11 2.54 ± 0.21 a 2.02 ± 0.13 a Oleate 1 m M 5.46 ± 0.31 4.70 ± 0.11 4.30 ± 0.25 b Significant differences from controls are a P < 0.002; b P < 0.03. Fig.3. EffectofHePCon[methyl- 14 C]choline incorporation into lipids in HepG2 cells. Pro- liferating cells were incubated for 6 h in EMEM/10% fetal bovine serum containing [methyl- 14 C]choline (60 l M ,55CiÆ mol )1 ) and different concentrations of HePC. The incorporation of [ 14 C]choline into cellular lipids and soluble intermediates of the CDP– choline pathway was determined, as described in the Materials and methods section. Results are expressed as means ± SEM for four independent samples in duplicate. Significant differences from controls are *P < 0.02; **P < 0.005. 4652 J. M. Jime ´ nez-Lo ´ pez et al.(Eur. J. Biochem. 269) Ó FEBS 2002 total enzyme activity in each dish. The results in Fig. 4 indicate that HePC caused a dose-dependent increase in cytosolic CT activity and that this was accompanied by a concomitant decrease in membrane-bound CT activity, suggesting that it acts on PC biosynthesis by modulating CT translocation between the membrane and the cytosol. It must be considered that the subcellular fractionation procedure could produce an alteration on the intracellular distribution of this enzyme. Thence, in order to corroborate the results above, we carried out the analysis of this enzyme activity in a homogenate from control and HePC-treated cells, demonstrating again that the total CT activity was unaltered by APC whilst the measure in the absence of liposomes, i.e. membrane-bound enzyme activity showed again a significant diminution in treated cells (i.e. 1.57 ± 0.06 nmolÆmin )1 Æmg protein )1 in control cells vs. 1.22 ± 0.08 nmolÆmin )1 Æmg protein )1 in HePC-treated cells, n ¼ 3; *P < 0.05). One possibility we must also bear in mind is that HePC might inhibit CT at the membrane level. To test this hypothesis we incubated in vitro the corresponding subcel- lular fraction in the presence of 50 l M and 100 l M HePC and analysed the activity of the enzymes in the CDP–choline pathway. We found that HePC did not in fact affect CK or CPT, neither did it alter particulate CT activity (results not shown). Nevertheless, as other authors have reported that the effects of HePC can be modulated by the quantity of lipid activator [15], we checked cytosolic CT in the presence of different amounts of PC/oleate liposomes. As can be seen in Fig. 5, the analysis of CT activity in the absence of liposomes demonstrates the existence in HepG2 cells of an active form of CT in the cytosol, which may correspond to the H-form previously described in this cell line [25]. However, CT activity was about five times higher in the presence of liposomes than in their absence. Cytosolic CT activity was inhibited by HePC in the presence of activating-lipid levels equal to or lower than 100 l M , although the degree of inhibition diminished concomitantly with an increase in the quantity of liposomes in the assay mixture (Fig. 5). Surprisingly, in the absence of PC/oleate liposomes, the addition of 100 l M HePC mark- edly increased soluble CT activity. HePC is a nonbilayer- forming lipid and so in the assay mixture it must be as micelles [10], which could act as a system for reconstituting inactive soluble CT, thus increasing the enzyme activity. DISCUSSION It has recently been shown both in vitro and in vivo that HePC exerts an antineoplastic effect [1,26], especially in the topical treatment of skin metastases of human mammary carcinoma [2,5], but to our knowledge no specific studies Fig. 5. Influence of HePC in vitro on cytosolic CTP:phosphocholine cytidylyltransferase activity in HepG2 cells. Cells growing in log-phase were harvested, sonicated briefly, and the cytosolic fraction obtained by centrifugation. Cytidylyltransferase activity was assayed in the absence or presence of 100 l M HePC and different concentrations of PC/oleate liposomes (molar ratio 1 : 1). Results are expressed as means ± SEM for three independent samples in duplicate. Significant differences from controls are *P <0.05;**P < 0.01. Fig. 4. Influence of HePC treatment on CTP:phosphocholine cytidylyltransferase activity in HepG2 cells. Cells growing in log-phase were incubated for 6 h with 50 l M or 100 l M HePC, or no additive (controls). Cytidylyltransferase (CT) activity was assayed both in the cytosolic supernatant and the particulate pellet. Data are expressed as percentages of distribution of cytosolic or membrane-bound CT activities per dish (3 · 10 6 cells). Results are expressed as means ± SEM for three independent samples in duplicate. Significant differences from controls are *P <0.04;**P < 0.001. Table 2. Influence of HePC treatment on enzyme activities in the CDP– choline pathway. Cells growing in log-phase were incubated for 6 h with 50 l M HePC whilst untreated cells were used as control. The different enzyme activities in the cytosol and particulate fractions [choline kinase (CK); CTP:phosphocholine cytidylyltransferase (CT) and diacylglycerol cholinephosphotransferase (CPT)] were determined as described in the Materials and methods section. Results are expressed as means ± SEM for three independent samples in dupli- cate. Significant difference from controls is *P <0.03. nmolÆmin )1 Æmg protein )1 Enzyme activity Control 50 l M HePC treatment CK 6.22 ± 0.10 5.63 ± 0.23 CT (cytosol) 9.57 ± 0.28 10.4 ± 0.97 CT (membranes) 1.82 ± 0.10 1.32 ± 0.09 a CPT 1.49 ± 0.07 1.51 ± 0.04 Ó FEBS 2002 Hexadecylphosphocholine and HepG2 cells (Eur. J. Biochem. 269) 4653 have been made into its effects upon hepatic cell lines. Thus we have examined the action of HePC, as a representative of the APC group, on the hepatoma cell line HepG2. Firstly, we evaluated the dose- and time-dependent cytotoxic and cytostatic activity of HePC against HepG2 cells, proving that concentrations of more than 100 l M for 6 h produce alterations in plasma membrane permeability and thence a rapid and unspecific detergent-like lytic effect. Nevertheless, at concentrations of less than 100 l M we found a reduction in viable cell numbers with no significant signs of toxicity. The quantities of HePC required to produce this antipro- liferative action agree with those encountered in MDCK [13], HeLa [27] and other neoplastic cell lines [15], indicating that HepG2 cells are moderately sensitive to the toxic and cytostatic activity of HePC concentrations in the lmolar range. Little is known about the biochemical mechanisms by which HePC, and probably also other lipid analogues, mediate their antiproliferative activity. These compounds are directly absorbed into both plasma and intracellular membranes, where they accumulate [28]. It might be assumed therefore that they interfere with membrane lipid composition and metabolic processes, although the effects of HePC on cell proliferation and metabolism may well differ depending upon the cell type or line [29] and/or the uptake rate into the cell [30]. Nevertheless, one consistent finding is that HePC causes a reduction in the biosynthesis of PC. In a similar way, our results demonstrate that in the tumoral hepatic cell line HepG2, HePC hinders the incorporation of radiolabelled choline into PC, the end- product of the CDP–choline pathway. This reduction in the incorporation of choline was not due to any alteration in its uptake by the cells, a finding which agrees with that of Geilen et al. [31] working with MDCK cells, but it does go against observations made by other researchers working with neuronal cells [16] and KB and Raji cells [32], in which the authors showed that APC did in fact interfere with the uptake of choline into the cell, leading to a decrease in PC biosynthesis. In the present work we exposed the cells to HePC between 5 min and 6 h and did not observe any change in the uptake of choline, thus demonstrating that in this cell line at least the mechanism via which HePC acts is unrelated to the availability of intracellular choline. Our results also indicate that the lower radioactivity in PC from exogenous choline compared to control cells was not due to any effect that HePC might have had upon the degradation of cellular PC or its secretion into the medium. These data contrast with those obtained with other cell lines such as Raji and KB cells, in which a pronounced decrease in the incorporation of [ 14 C]choline into PC was paralleled by an increase in the degradation of this phospholipid [32]. It is worth emphasizing that the reduction we observed in the incorporation of exogenous choline into PC was accompanied by an accumulation of radiolabel in CP and a reduction in the radioactivity associated to CDP–choline, suggesting that some of the enzymes involved in the biosynthesis of PC must be the main target of HePC. Cytosolic CK and membrane-bound CPT remained unalterated by HePC whilst CT activity was significantly modified. It is well documented that CT is inactive in the cytosolic form but becomes active when bound to mem- branes, and therefore one possible mode of CT regulation would be via translocation between membranes and cytosol [33]. After 6 h exposure to 50 l M HePC, CT activity diminished by 20% in the particulate fraction of the cells and increased in the cytosolic fraction, as can be seen when the values are expressed as percentages of total CT activity. These results demonstrate that HePC affects only the distribution of CT and not its total activity. HePC therefore appears to act on the CT translocation mechanism. It is widely accepted that oleate stimulates PC synthesis in a number of cells and tissues, promoting the translocation of cytosolic CT to the membrane [34]. This prompted us to investigate the combined effect of oleate and HePC on PC synthesis. The results obtained demonstrate that the effect of HePC is clearly buffered by oleate suggesting that this fatty acid and HePC act in an opposite way. As oleate favours the translocation of CT to membrane our results corroborate the interference of HePC on this regulatory process. Thence, HePC could act on CT activity either by promoting the release of the enzyme from the membrane to the cytosol or by hindering the insertion of the cytosolic form into the membrane. To explore these possibilities we carried out experiments in vitro to analyse the effect of HePC on CT activity in the cytosol and in the particulate fraction isolated from HepG2 cells. With regard to cytosolic CT, we looked into the effect of HePC in the presence of different amounts of PC/oleate vesicles as lipid activators. In the presence of low-levels of activating lipid, the soluble activity was significantly reduced, suggesting that HePC interferes with the translo- cation of the soluble form of the enzyme to the lipid vesicles. This inhibitory effect was not observed in the presence of high concentrations of PC/oleate liposomes as HePC acts as a competitive inhibitor of the lipid activator [15]. 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Ó FEBS 2002 Hexadecylphosphocholine and HepG2 cells (Eur. J. Biochem. 269) 4655 . effect on the biosynthesis of PC in HepG2 cells at the level of CT activity. It interferes with the translocation process of CT from the cytosol to the membrane. exclusion assay and LDH release into the medium. Inhibition of PC biosynthesis by HePC We examined the effect of HePC on the biosynthesis of PC by using