Gender-associated differences in oral microbiota and salivary biochemical parameters in response to feeding.

Saliva plays a key role in food absorption and digestion mainly due to both its enzymes and microbiota. The main objective of this study was to compare the oral microbiota and salivary parameters between men and women in response to feeding. To answer this question, we set up a pilot study on 10 male and 10 female subjects to examine the role of saliva in glycaemia physiology. Biological parameters and the microbiotal composition of saliva were analyzed in fasted and fed states. The results show that the level of blood glucose was not different between men and women in the fasted state (88.00 mg/dL ± 6.38 vs 87.00 mg/dL ±8.07, p = 0.9149) or in the fed state (102.44 mg/dL ± 14.03 vs 116.9 mg/dL ± 25, p = 0.1362). Free fatty acids (FFA 0.15 mmol/L ± 0.15 vs 0.07 mmol/L ± 0.07, p = 0,0078), cholesterol (0.53 mmol/L ± 0.30 vs 0.15 mmol/L ± 0.14, p < 0.0001), and total saliva proteins (13.2 g/L ± 4.31 vs 9.02 g/L ± 6.98, p = 0.0168) were decreased after feeding, as well as the saliva lipase (27.89 U/L ± 25.7 vs 12.28 U/L ± 4.85, p = 0.0126). A very significant increase in the relative abundance of Streptococcaceae (24.56 ± 9.32 vs 13.53 ± 7.47, p = 0.00055) and a decrease in Prevotellaceae (34.45 ± 9.30 vs 17.43 ± 9.03, p = 0.00055) were observed in the fed condition. When investigating gender-related differences in the fasted state, men showed higher levels of cholesterol (0.71 mmol/L ± 0.26 vs 0.40 mmol/L ± 0.27, p = 0.0329), FFA (0.25 mmol/L ± 0.18 vs 0.08 mmol/L ± 0.06, p = 0.0049), and triglycerides (0.24 mmol/L ± 0.15 vs 0.09 mmol/L ± 0.04, p = 0.006) than women. Finally, differences could be observed in saliva microbiota between men and women in the fasted condition but even more in the fed condition, where Porphyromonas and Capnocytophaga were overrepresented in the male salivary samples compared with female saliva. Thus, biological parameters and microbiota in saliva could be the signatures of the feeding conditions and sex gender status.

Analysis of hydrophilic and lipophilic choline compounds in radioresistant and radiosensitive glioblastoma cell lines by HILIC-ESI-MS/MS.

A new method based on hydrophilic interaction chromatography-electrospray ionisation-tandem mass spectrometry (HILIC-ESI-MS/MS) coupled to the use of a stable isotope labelled substrate was developed to study the metabolism of choline (Cho) compounds in two human glioblastoma multiform (GBM) cell lines with different responses to ionising radiation. Analysis was performed in the positive ion mode using multiple reaction monitoring. This fast, sensitive and selective method enabled the profiling of both hydrophilic and lipophilic Cho-containing compounds, to analyse specifically different phosphatidylcholine (PtdCho) molecular species, and to measure simultaneously native and labelled Cho metabolites. Radioresistant (SF763) and radiosensitive (SF767) cells were incubated for 8 h with d(9)-Cho. Higher native Cho and phosphocholine (PCho) concentrations and higher uptake of d(9)-Cho and formation of d(9)-PCho were found in the radioresistant cell line. The similar low concentrations of native cytidine 5'-diphosphocholine (CDP-Cho) and d(9)-CDP-Cho in both cell lines show that CDP-Cho is the limiting metabolite in the two models. The turnovers (percentage of each d(9)-Cho compound in its respective pool, i.e. native + labelled) were lower in radioresistant cells for all Cho compounds, suggesting a global PtdCho metabolism more active in radiosensitive cells that could be related to their higher proliferation rate.

Ligands of the antiestrogen-binding site induce active cell death and autophagy in human breast cancer cells through the modulation of cholesterol metabolism.

We have recently reported that cytostatic concentrations of the microsomal antiestrogen-binding site (AEBS) ligands, such as PBPE (N-pyrrolidino-(phenylmethyphenoxy)-ethanamine,HCl) and tamoxifen, induced differentiation characteristics in breast cancer cells through the accumulation of post-lanosterol intermediates of cholesterol biosynthesis. We show here that exposure of MCF-7 (human breast adenocarcinoma cell line) cells to higher concentrations of AEBS ligands triggered active cell death and macroautophagy. Apoptosis was characterized by Annexin V binding, chromatin condensation, DNA laddering and disruption of the mitochondrial functions. We determined that cell death was sterol- and reactive oxygen species-dependent and was prevented by the antioxidant vitamin E. Macroautophagy was characterized by the accumulation of autophagic vacuoles, an increase in the expression of Beclin-1 and the stimulation of autophagic flux. We established that macroautophagy was sterol- and Beclin-1-dependent and was associated with cell survival rather than with cytotoxicity, as blockage of macroautophagy sensitized cells to AEBS ligands. These results show that the accumulation of sterols by AEBS ligands in MCF-7 cells induces apoptosis and macroautophagy. Collectively, these data support a therapeutic potential for selective AEBS ligands in breast cancer management and shows a mechanism that explains the induction of autophagy in MCF-7 cells by tamoxifen and other selective estrogen receptor modulators.

Cell surface adenylate kinase activity regulates the F(1)-ATPase/P2Y (13)-mediated HDL endocytosis pathway on human hepatocytes.

We have previously demonstrated on human hepatocytes that apolipoprotein A-I binding to an ecto-F(1)-ATPase stimulates the production of extracellular ADP that activates a P2Y(13)-mediated high-density lipoprotein (HDL) endocytosis pathway. Therefore, we investigated the mechanisms controlling the extracellular ATP/ADP level in hepatic cell lines and primary cultures to determine their impact on HDL endocytosis. Here we show that addition of ADP to the cell culture medium induced extracellular ATP production that was due to adenylate kinase [see text] and nucleoside diphosphokinase [see text] activities, but not to ATP synthase activity. We further observed that in vitro modulation of both ecto-NDPK and AK activities could regulate the ADP-dependent HDL endocytosis. But interestingly, only AK appeared to naturally participate in the pathway by consuming the ADP generated by the ecto-F(1)-ATPase. Thus controlling the extracellular ADP level is a potential target for reverse cholesterol transport regulation.

The nucleotide receptor P2Y13 is a key regulator of hepatic high-density lipoprotein (HDL) endocytosis.

Cell surface receptors for high-density lipoprotein (HDL) on hepatocytes are major partners in the regulation of cholesterol homeostasis. We recently identified a cell surface ATP synthase as a high-affinity receptor for HDL apolipoprotein A-I (apoA-I) on human hepatocytes. Stimulation of this ectopic ATP synthase by apoA-I triggered a low-affinity-receptor-dependent HDL endocytosis by a mechanism strictly related to the generation of ADP. This suggests that nucleotide G-protein-coupled receptors of the P2Y family are molecular components in this pathway. Only P2Y1 and P2Y13 are present on the membrane of hepatocytes. Using both a pharmacological approach and small interference RNA, we identified P2Y13 as the main partner in hepatic HDL endocytosis, in cultured cells as well as in situ in perfused mouse livers. We also found a new important action of the antithrombotic agent AR-C69931MX as a strong activator of P2Y13-mediated HDL endocytosis.

New insight on the molecular mechanisms of high-density lipoprotein cellular interactions.

High-density lipoprotein (HDL) cholesterol is an independent negative risk factor for coronary artery disease and thus represents today the only protective factor against atherosclerosis. The protective effect of HDL is mostly attributed to its central function in reverse cholesterol transport (RCT), a process whereby excess cell cholesterol is taken up and processed in HDL particles, and is later delivered to the liver for further metabolism and bile excretion. This process relies on specific interactions between HDL particles and cells, both peripheral (cholesterol efflux) and hepatic (cholesterol disposal) cells, and on the maturation of HDL particles within the vascular compartment. The plasma level of HDL cholesterol will thus result also from the complex interplay with cellular partners. Among them, some contribute to HDL formation - for instance ATP binding cassette AI protein - while others are mostly involved in HDL catabolism, the scavenger receptor-class B type I or the recently described membrane-bound ATP synthase/hydrolase. The last decade has seen major breakthroughs in the identification and elucidation of the role of cellular partners of HDL metabolism, and in their transcriptional regulations, opening up new perspectives in the modulation of HDL cholesterol.

Sulforaphane, a naturally occurring isothiocyanate, induces cell cycle arrest and apoptosis in HT29 human colon cancer cells.

Sulforaphane is an isothiocyanate that is present naturally in widely consumed vegetables and has a particularly high concentration in broccoli. This compound has been shown to block the formation of tumors initiated by chemicals in the rat. Although sulforaphane has been proposed to modulate the metabolism of carcinogens, its mechanism of action remains poorly understood. We have previously demonstrated that sulforaphane inhibits the reinitiation of growth and decreases the cellular viability of quiescent human colon carcinoma cells (HT29). Moreover, the weak effect observed on differentiated CaCo2 cells suggests a specific anticancer activity for this compound. Here we investigated the effect of sulforaphane on the growth and viability of HT29 cells during their exponentially growing phase. We observed that sulforaphane induced a cell cycle arrest in a dose-dependent manner, followed by cell death. This sulforaphane-induced cell cycle arrest was correlated with an increased expression of cyclins A and B1. Moreover, we clearly demonstrated that sulforaphane induced cell death via an apoptotic process. Indeed, a large proportion of treated cells display the following: (a) translocation of phosphatidylserine from the inner layer to the outer layer of the plasma membrane; (b) typical chromatin condensation; and (c) ultrastructural modifications related to apoptotic cell death. We also showed that the expression of p53 was not changed in sulforaphane-treated cells. In contrast, whereas bcl-2 was not detected, we observed increased expression of the proapoptotic protein bax, the release of cytochrome c from the mitochondria to the cytosol, and the proteolytic cleavage of poly(ADP-ribose) polymerase. In conclusion, our results strongly suggest that in addition to the activation of detoxifying enzymes, induction of apoptosis is also involved in the sulforaphane-associated chemoprevention of cancer.

Characterization of two high-density lipoprotein binding sites on porcine hepatocyte plasma membranes: contribution of scavenger receptor class B type I (SR-BI) to the low-affinity component.

Two HDL(3) high- and low-affinity binding sites are present on the human hepatoma cell line (HepG(2)). Recently, we have suggested that the high-affinity binding sites might modulate the endocytosis of HDL through the low-affinity binding sites [Guendouzi, K. (1998) Biochemistry 37, 14974-14980], highlighting the physiological importance of this family of HDL high-affinity binding sites. The present data demonstrate the presence of HDL(3) high-affinity (K(d) = 0.37 microg/mL, B(max) = 260 ng/mg of protein) and low-affinity (K(d) = 86.2 microg/mL, B(max) = 14 300 ng/mg of protein) binding sites on purified porcine hepatocyte plasma membranes. By contrast, free apoA-I was strictly specific to the high-affinity sites (K(d) = 0.2 microg/mL and B(max) = 72 ng/mg of protein). Competition experiments between (125)I-labeled HDL(3) and either LDL, oxidized LDL, or anti-SR-BI IgG as competitors show that SR-BI is mostly responsible (70% displacement) for the binding of HDL(3) to the low-affinity binding sites. By contrast, the same competition experiments using (125)I-labeled free apoA-I clearly excluded SR-BI as the high-affinity binding receptor. We conclude that the binding of HDL onto hepatocyte plasma membranes involves: (1) two low-affinity binding receptors, one being SR-BI; (2) one family of high-affinity binding sites unrelated to SR-BI.

Identification of an ApoA-I ligand domain that interacts with high-affinity binding sites on HepG2 cells.

We have previously described the presence of two (high- and low-affinity) HDL binding sites on the hepatoma cell line (HepG2) (R. Barbaras, X. Collet, H. Chap, and B. Perret (1994) Biochemistry 33, 2335-2340]. Moreover, apoA-I, the major HDL apolipoprotein, interacts with these two binding sites, while lipid-free apoA-I binds only to the high-affinity sites. Using tryptic HDL fragments and HepG2 cell monolayers as an "affinity matrix," we identified an apoA-I peptide of 16 amino acids, spanning between residues 62 and 77, as a ligand domain. The corresponding synthetic peptide displays high-affinity (K(d) approximately 10(-7) M) and low-capacity (B(max) 8 pmol/mg of cell protein) binding components. Competition experiments with this peptide, using (125)I-labeled free apoA-I as a ligand, show that this binding corresponds to the high-affinity binding sites already described. In conclusion, we identified the apoA-I 62-77 region as a specific high-affinity ligand domain of HDL on HepG2 cells.

Competitive inhibition of choline phosphotransferase by geranylgeraniol and farnesol inhibits phosphatidylcholine synthesis and induces apoptosis in human lung adenocarcinoma A549 cells.

We have previously shown that, among various isoprenoids, farnesol and geranylgeraniol specifically induced actin fiber disorganization, growth inhibition, and apoptosis in human lung adenocarcinoma A549 cells (Miquel, K., Pradines, A., and Favre, G. (1996) Biochem. Biophys. Res. Commun. 225, 869-876). Here we demonstrate that isoprenoid-induced apoptosis was preceded by an arrest in G0/G1 phase. The isoprenoid effects were independent of protein prenylation and of mitogen-activated protein kinase activity. Moreover, geranylgeraniol and farnesol induced a rapid inhibition of phosphatidylcholine biosynthesis at the last step of the CDP-choline pathway controlled by choline phosphotransferase and not at the level of CTP:phosphocholine cytidylyltransferase, the key enzyme of the pathway. Inhibition of choline phosphotransferase was confirmed by in vitro assays on microsomal fractions, which clearly showed that the isoprenoids acted by competitive inhibition with the diacylglycerol binding. Exogenous phosphatidylcholine addition prevented all the biological effects of the isoprenoids, including actin fiber disorganization and apoptosis, suggesting that inhibition of phosphatidylcholine biosynthesis might be the primary event of the isoprenoid action. These data demonstrate the molecular mechanism of geranylgeraniol and farnesol effects and suggest that the mevalonate pathway, leading notably to prenylated proteins, might be linked to the control of cell proliferation through the regulation of phosphatidylcholine biosynthesis.

A genetic defect in phosphatidylcholine biosynthesis triggers apoptosis in Chinese hamster ovary cells.

We have investigated the cell death of a Chinese hamster ovary mutant (MT-58) with a thermo-sensitive CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme of the CDP-choline pathway for phosphatidylcholine biosynthesis (Esko, J. D., Wermuth, M. M., and Raetz, C. R. H. (1981) J. Biol. Chem. 256, 7388-7393). After MT-58 cells were shifted to the restrictive temperature of 40 degrees C, the cytidylyltransferase was inactivated immediately leading to a decrease in phosphatidylcholine biosynthesis and cell death. DNA content and number of cells in the S phase decreased significantly in the dying MT-58 cells according to flow cytometrical analyses. The fragmentation of genomic DNA was detected by DNA ladders in agarose gel and release of the prelabeled genomic DNA into cytosolic fractions 14 h after the temperature shift. The dying cells underwent a dramatic reduction of cellular volume while maintaining the membrane containment of cellular contents. These events indicated that the inactivation of cytidylyltransferase triggered apoptosis in Chinese hamster ovary cells. This is the first report that apoptosis was induced in cultured cells, not by an added agent, but by a mutation in phospholipid biosynthesis.

Phosphatidylcholine turnover in activated human neutrophils. Agonist-induced cytidylyltransferase translocation is subsequent to phospholipase D activation.

Phosphatidylcholine synthesis and degradation are tightly regulated to assure a constant amount of the phospholipid in cellular membranes. The chemotactic peptide fMLP and the phorbol ester, phorbol 12-myristate 13-acetate, are known to stimulate phosphatidylcholine degradation by phospholipase D in human neutrophils. fMLP alone triggered phosphatidylcholine breakdown into phosphatidic acid, but did not stimulate phosphatidylcholine synthesis or activation of the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase. Adding cytochalasin B to fMLP led to some conversion of phosphatidic acid into diglyceride, and fMLP was then able to trigger choline incorporation into phosphatidylcholine, and cytidylyltransferase translocation from cytosol to membranes. Inhibition of phosphatidyl-choline-phospholipase D activation with tyrphostin led to inhibition of choline incorporation. Therefore, phosphatidic acid-derived diglyceride but not phosphatidic acid alone was effective to promote cytidylyltransferase translocation. With phorbol 12-myristate 13-acetate as agonist, and by selective labeling of phosphatidylinositol and phosphatidylcholine, we demonstrated that only phosphatidylcholine-derived diglyceride participated in cytidylyltransferase translocation. Oleic acid stimulated phosphatidylcholine synthesis, but induced a weak increase in diglyceride and a slight cytidylyltransferase translocation, and did not stimulate phospholipase D activity. Our data established that only diglyceride derived from phosphatidylcholine degradation by the phospholipase D/phosphatidate phosphatase pathway are required for agonist-induced cytidylyltransferase translocation and subsequent choline incorporation into phosphatidylcholine.

Requirement of phosphatidylcholine for normal progression through the cell cycle in C3H/10T1/2 fibroblasts.

We have investigated the possible requirement of phosphatidylcholine for normal progression through the cell cycle of C3H/10T1/2 fibroblasts. Incubation of the cells in a medium with 0.5% serum synchronized the cells in the G0 stage of the cell cycle. Supplementation of the cells with 10% dialyzed and delipidated serum +/- choline resulted in normal cell division and growth for cells with choline, whereas cell division was markedly impaired in the absence of choline. Flow cytofluorometry analysis indicated that after 4 days in the absence of choline, 85% of the fibroblasts were in the G1 phase. Addition of choline resulted in synchronous synthesis of DNA with a peak occurring after 14 h. Incubation of cells with 0.5% serum had no effect on phosphatidylcholine (PC) levels in cells supplemented with 28 microM choline, but the concentration of PC was reduced from 32 to 20 nmol/10(6) cells after 1 day of incubation in the absence of choline. Supplementation with dialyzed serum, but not dialyzed and delipidated serum, allowed choline-deficient cells to replicate normally. This was attributed to the presence of lysophosphatidylcholine in dialyzed serum as this lipid, but not other lipids (e.g., phosphatidylcholine or mitogenic lipids) was able to replace the choline requirement. The choline-deficient effect was not complete; some DNA synthesis occurred in the absence of choline in the medium, and approximately 30% of the cells completed mitosis in 35 h compared to 100% in the presence of choline. The data suggest that phosphatidylcholine is required for normal progression of the cell cycle beyond the G1 phase and is unrelated to the induction of G0 to G1 transition. Choline deficiency should be a useful method for synchronizing cells in the G1 phase.

Phorbol myristate acetate stimulates [3H]choline incorporation into phosphatidylcholine independently of the 'de novo' pathway in Krebs-II ascitic cells: a unique effect of phorbol ester on choline uptake.

The effect of phorbol 12-myristate 13-acetate (PMA) on [3H]choline incorporation into phosphatidylcholine (PtdCho) and on the 'de novo' pathway of PtdCho synthesis has been investigated, compared with that of oleic acid, in ascitic-strain Krebs-II cells. Both compounds stimulated [3H]choline incorporation into PtdCho, but the PMA-induced incorporation was saturable at concentrations of the agonist around 100 nM, whereas no saturation was noticed with oleic acid up to 1 mM. Chase experiments showed no effect of PMA on the conversion of phosphocholine into CDP-choline. The phorbol ester did not stimulate any of the enzyme activities of the 'de novo' pathway, whereas oleic acid increased specifically by 2.5-fold the CTP:phosphocholine cytidylyltransferase (CT, EC 2.7.7.15) activity. In addition, no change in the subcellular distribution of CT was observed upon incubation with PMA, in contrast with oleic acid treatment. Cells challenged with oleic acid showed a 25-fold increase in diradylglycerol (DG) content, which was not modified upon incubation with 200 nM PMA, the most effective concentration of phorbol ester promoting choline incorporation. Subcellular fractionation of Krebs-II cells on Percoll gradients revealed that [3H]PMA and 1-radyl-2-[3H]oleoyl-glycerol, derived from exogenously supplied [3H]oleic acid, both exhibited the same enrichment in the endoplasmic reticulum. We have previously shown that the labelled fatty acid also accumulated in the endoplasmic reticulum [Tercé, Record, Tronchère, Ribbes and Chap (1992) Biochem. J. 282, 333-338]. However, PMA induced a stimulation of choline uptake, which was not provoked by PMA 4-O-methyl ether, which interacts poorly with protein kinase C. Our data provide evidence that the enhancement of [3H]choline incorporation into PtdCho triggered by PMA and oleic acid proceeds via completely distinct mechanism(s).

Reversible translocation of cytidylyltransferase between cytosol and endoplasmic reticulum occurs within minutes in whole cells.

Addition of oleic acid to Krebs II cells induced a rapid incorporation of [3H]choline into phosphatidylcholine, since 500 microM of the fatty acid stimulated choline incorporation by 5-fold over the control after 5 min of incubation. In fact, a noticeable increase in phosphatidylcholine labelling could be monitored immediately after 1 min of cell incubation with [3H]choline, at which time 50% of cytosolic cytidylyltransferase activity (EC 2.7.7.15), the regulatory enzyme of phosphatidylcholine synthesis, was translocated on to membranes. Non-esterified [3H]oleic acid content was also increased in the same range of time in the particulate fraction. Subcellular fractionation indicated that endoplasmic reticulum was the unique binding site for cytidylyltransferase even after 1 min of incubation. Also, [3H]oleic acid accumulated mainly in the same internal membrane. Addition of exogenous albumin to cells prelabelled with [3H]oleic acid induced the release of 50% of membrane-bound cytidylyltransferase activity within 1 min, together with a decrease in unesterified oleic acid in the same membrane. Although total depletion of oleic acid was obtained, total release of membrane-bound cytidylyltransferase was not. The remaining minor pool of membrane-bound cytidylyltransferase was not affected by cell incubation with dibutyryl cyclic AMP, suggesting that this pool was neither regulated by fatty acid nor modulated by cyclic-AMP-dependent protein phosphorylation. Addition of [3H]oleic acid directly to an homogenate led to a less specific accumulation of the fatty acid in the endoplasmic reticulum, but cytidylyltransferase remained exclusively associated with this membrane. We concluded that in vivo translocation of cytidylyltransferase provoked by oleic acid concerns one specific pool of the enzyme distinct from the enzyme firmly bound to endoplasmic reticulum, but other factor(s) than fatty acid seem to be required to explain the specificity of endoplasmic reticulum for cytidylyltransferase binding.

Subcellular localization of enzymes related to PAF metabolism in Krebs-II ascites cells: evidence for the lack of intracellular PAF transfer activity.

We have investigated various steps in the metabolism of Platelet-Activating Factor (PAF-acether or PAF) at the subcellular level in Krebs-II ascites cells. Microsomes contained an active acetyltransferase located on a heavy-rough domain of the endoplasmic reticulum quite rich in ribosomes, as monitored by [3H]uridine labelling, and which displayed a very high density across the Percoll gradient. This membrane domain, which was separated from all other cellular organelles including peroxisomes, also contained a membrane-bound acetylhydrolase with similar activity as the acetyltransferase. However most part of the cellular acetylhydrolase was located in the cytosol, which was actually devoid of PAF transfer activity, normally involved in transport of the mediator within the cell.

Cytidylyltransferase translocation onto endoplasmic reticulum and increased de novo synthesis without phosphatidylcholine accumulation in Krebs-II ascite cells.

Addition of oleic acid to Krebs-II cells stimulated by 9-fold [3H]choline incorporation into choline glycerophospholipids without affecting the selective incorporation of the precursor into diacyl subclass (90% of total [3H]choline glycerophospholipids). The total activity of cytidylyltransferase (E.C. 2.7.7.15), the regulatory enzyme of choline glycerophospholipid synthesis, was increased in the particulate fraction at the expense of cytosol. Free [3H]oleic acid was also associated with the particulate fraction. Subcellular fractionation of membranes on Percoll gradient, indicated that the endoplasmic reticulum, which contained 90% of total cell free oleic acid, was the unique target for the translocation of cytidylyltransferase. [3H]oleic acid was incorporated almost exclusively into phosphatidylcholine and corresponded to a synthesis of 9 nmol/h per 10(6) cells. Based on [3H]choline incorporation a net synthesis of 22 nmol/h per 10(6) cells was determined. However, oleic acid treatment did not change the total amount of phosphatidylcholine (45 nmol/10(6) cells) and other phospholipids; also no modification in the subcellular distribution of phospholipids was observed. It is concluded that the stimulation of the de novo synthesis of phosphatidylcholine which involves translocation of cytidylyltransferase onto the endoplasmic reticulum, is accompanied by a renewal of their polar head group. Also exogenous oleic acid induces an enhanced fatty acid turnover, highly specific for phosphatidylcholine. Therefore, Krebs-II cells exhibited a high degree of regulation of their phosphatidylcholine content, suggesting a parallel stimulation of both synthesis and degradation.

Modulation of CTP:phosphocholine cytidylyltransferase translocation by oleic acid and the antitumoral alkylphospholipid in HL-60 cells.

Short time effect of oleate and 1-O-alkyl-2-O-methyl-rac-glycero-3-phosphocholine (AMGPC) on choline incorporation into phosphatidylcholines were studied in HL-60 cells. The non lytic concentration of 50 microM oleate induced a three-fold increase in [3H]choline incorporation into phosphatidylcholine. This stimulation was accompanied by a translocation of the CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) from cytosol to membranes. By contrast, the ether-lipid AMGPC inhibited [3H]choline incorporation into phosphatidylcholine by 60% at 10 microM. AMGPC had no effect on choline kinase or choline phosphotransferase activities. When AMGPC was added separately to an homogenate, a particulate or a cytosolic fraction, cytidylyltransferase inhibition was observed only in the homogenate. However on particulates recovered from homogenates treated with increasing concentrations of AMGPC, membranous cytidylyltransferase activity decreased dose-dependently. Thus AMGPC had no effect on cytidylyltransferase activity itself but inhibited its translocation from cytosol to membrane. At variance with the well-established positive effect on cytidylyltransferase translocation induced by fatty acids, this is the first demonstration that AMGPC can inhibit cytidylyltransferase translocation in cell-free system.

PAF-acether transfer activity in HL-60 cells is induced during differentiation.

Platelet-activating factor is a proinflammatory lipid active at subnanomolar concentrations. The intermembrane transfer of a biologically active PAF analog has been previously demonstrated in macrophages. Here we demonstrate that the specific activity of this transfer activity increases when HL-60 cells are induced to differentiate by treatment with dimethyl sulfoxide, dibutyryl cAMP or phorbol diester. In undifferentiated HL-60 cells, methylcarbamyl-PAF transfer activity was only 0.56 U.min-1.mg-1. This basal value was increased 2.6 and 6.7 times upon granulocytic and macrophagic differentiation, respectively. On the other hand, the transfer of 2-O-methyl-PAF, a cytotoxic analog with no PAF biological activity, remained very low and did not vary during differentiation.