©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Lysophosphatidylcholine and 1-O-Octadecyl-2-O-Methyl-rac- Glycero-3-Phosphocholine Inhibit the CDP-Choline Pathway of Phosphatidylcholine Synthesis at the CTP:Phosphocholine Cytidylyltransferase Step (*)

(Received for publication, October 31, 1994; and in revised form, December 27, 1994)

Kevin P. Boggs (1) Charles O. Rock (1) (2) Suzanne Jackowski (1) (2)(§)

From the  (1)Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 38101 and the (2)Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The regulation of the CDP-choline pathway of phosphatidylcholine synthesis at the CTP:phosphocholine cytidylyltransferase (CT) step by lysophosphatidylcholine (LPC) and the nonhydrolyzable LPC analog, 1O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH(3)), was investigated in a colony-stimulating factor 1-dependent murine macrophage cell line. LPC inhibited phosphatidylcholine synthesis in vivo and led to the accumulation of choline and phosphocholine coupled to the disappearance of CDP-choline pointing to CT as the intracellular target. LPC neither inhibited cell growth nor decreased the cellular content of CT or altered the distribution of CT between soluble and particulate subcellular fractions. The inhibition of phosphatidylcholine synthesis was specific for LPC since lysophospholipids lacking the choline headgroup were not inhibitors. ET-18-OCH(3) was a more potent inhibitor of phosphatidylcholine synthesis than LPC and caused the translocation of CT from the soluble compartment to the particulate compartment. Both LPC and ET-18-OCH(3) were inhibitors of CT activity in vitro and kinetic analysis showed competitive inhibition with respect to the lipid activator. These data point to LPC as a negative regulator of de novo phosphatidylcholine synthesis that acts at the CT step and establish the mechanism for the inhibition of phosphatidylcholine biosynthesis by antineoplastic phospholipids.


INTRODUCTION

PtdCho (^1)is a major structural building block of biological membranes and the precursor to second messengers following stimulation of cell surface receptors. CTP:phosphocholine cytidylyltransferase (CT), which catalyzes the formation of CDP-choline, has long been recognized as the key enzyme controlling the PtdCho biosynthetic pathway (for reviews, see Refs. 1 and 2) and plays a determinant role in regulating de novo membrane phospholipid accumulation (Fig. 1). In cultured eukaryotic cells, CT activity is necessary for cell survival(3, 4) , not only because PtdCho is itself a major membrane constituent, but PtdCho also serves as a precursor for two other abundant membrane phospholipids: sphingomyelin (5) and phosphatidylethanolamine(6) . CT absolutely requires the presence of specific lipids for activity(7, 8) and the protein is thought to be an intranuclear protein (9, 10, 11, 12) that is activated by interactions with membranes containing diacylglycerols (13, 14) , fatty acids(15) , or deficient in PtdCho(16, 17) . CT is phosphorylated at several sites in vivo(18, 19) . A role for phosphorylation in the regulation of CT activity is suggested by the finding that the soluble form of CT is highly phosphorylated whereas the particulate form is relatively dephosphorylated(10, 11, 20) . CT phosphorylation is a periodic cell cycle regulated event in a murine macrophage cell line, BAC1.2F5, and reduced CT activity correlates with increased phosphorylation of the protein(21) . The requirement for the CDP-choline pathway can be circumvented by providing cells with exogenous LPC(22, 23) , which is rapidly acylated to form PtdCho (Fig. 1).


Figure 1: Pathways for the biosynthesis of the major membrane phospholipids in cultured animal cells. Choline is taken up from the medium and phosphorylated by choline kinase (CK). CT converts phosphocholine to CDP-choline which is then utilized by CPT along with diacylglycerol to form PtdCho. The uptake and acylation of exogenous LPC circumvents the requirement for the CDP-choline pathway by the direct formation of PtdCho. PtdCho then serves as a precursor to both sphingomyelin and phosphatidylethanolamine via phosphatidylserine. PtdCho is also a precursor to LPC via the action of phospholipase A(2). LPC and ET-18-OCH(3) are proposed to inhibit the CDP-choline pathway at the CT step.



There is a growing interest in antineoplastic ether-linked phospholipid analogs since they do not directly target DNA and could potentially complement existing DNA-directed anticancer compounds. ET-18-OCH(3) is a non-metabolizable LPC analog and belongs to the first generation of ether lipids tested as a growth inhibitor(24) . There have been numerous studies demonstrating the selective cytotoxic action of ET-18-OCH(3) against transformed cells in whole animals and tissue culture(25, 26, 27, 28, 29, 30, 31, 32, 33) . Ongoing clinical trials are evaluating the effectiveness of different antineoplastic ether-linked phospholipid analogs against a variety of cancers (for review, see (34) ), and one example of encouraging clinical research with ET-18-OCH(3) is its use in purging leukemic bone marrow for autologous bone marrow transplantation(35) . In 1979, Modolell et al.(36) reported that ET-18-OCH(3)-treated cells have reduced levels of PtdCho. Subsequently, several groups showed that ET-18-OCH(3) decreases choline incorporation into PtdCho (37, 38, 39, 40) . The mechanism for inhibition of this pathway has not been established although ET-18-OCH(3) is reported to diminish the association of CT with the membrane(40) . A related analog, hexadecylphosphocholine, also inhibits choline incorporation into PtdCho (41, 42, 43) and partially inhibits CT activity when added to whole cell lysates(44) . Since LPC circumvents the requirement for de novo PtdCho biosynthesis via the CDP-choline pathway (45) and LPC analogs inhibit choline incorporation(37, 38, 39, 40, 41, 42, 43) , the goal of this research is to test the hypothesis that LPC and antineoplastic phospholipids negatively regulate the CDP-choline biosynthetic pathway at the CT step (Fig. 1) and to determine the mechanism of CT regulation by lysophospholipids.


EXPERIMENTAL PROCEDURES

Materials

Sources of supplies were: DuPont NEN, [P]orthophosphate (carrier free), phospho[^14C]choline (specific activity 50 mCi/mmol), [^3H]choline (specific activity 86.6 Ci/mmol); CDP-[^14C]choline (specific activity 58 mCi/mmol), [^3H]thymidine (specific activity 87.7 Ci/mmol), and I-protein A (specific activity 30 mCi/mg). Analtech, Silica Gel G thin-layer plates; Avanti Polar Lipids, egg yolk lysophosphatidylcholine (LPC); Hyclone Inc., fetal calf serum; Life Technologies, Inc., Dulbecco's modified Eagle's medium, choline-free Dulbecco's modified Eagle's medium, and other tissue culture supplies; Sigma, phenylmethylsulfonyl fluoride, CDP-choline, lactate dehydrogenase assay kit, digitonin, leupeptin, CTP, and aprotinin. Polyclonal rabbit antibodies against purified rat CT were the same as described by Jackowski(21) . Recombinant CSF-1 was kindly provided by Genetics Institute. All other materials were reagent-grade or better.

Cell Culture

The cell line used in this work was BAC1.2F5, a macrophage-like, CSF-1-dependent clone derived from the BAC1 line (46) which originated from transfection of splenic adherent cells from a (BALB/c times A.CA)F(1) mouse with simian virus 40 origin-defective DNA(47) . The BAC1.2F5 line exhibits many properties of macrophages, including production of lysozyme, collagenase, and esterase. BAC1.2F5 cells have an absolute requirement for CSF-1 for both proliferation and survival(48) . BAC1.2F5 cells were routinely grown in Dulbecco's modified Eagle's medium supplemented with 15% fetal calf serum, 25% L-cell conditioned medium, 1% glutamine, and 20 mM HEPES, pH 7.3. The human growth factor was as effective as murine CSF-1 and stimulation of the cells with 25% L-cell conditioned medium was equivalent to the addition of 300 ng/ml CSF-1.

Mitogenesis and Cell Growth

The effect of LPC and ET-18-OCH(3) on the mitogenic response of BAC1.2F5 cells to CSF-1 and long-term growth was determined using [^3H]thymidine incorporation and viable cell number determinations, respectively, as described(49) .

Isolation of CT from Endogenous Lipids

CT was purified from Sf9 insect cells infected with the bCT baculovirus(50) . After 48 h infection, cells were harvested by centrifugation and the pellet was lysed by incubation in lysis buffer (10 mM NaCl, 1 mM EDTA, 2 mM dithiothreitol, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin (0.16 trypsin inhibitor units/ml), 50 mM sodium fluoride, 100 µM sodium orthovanadate, 100 nM okadaic acid, 10 mM HEPES, pH 7.4) for 1 h on ice. The cells were disrupted by sonication and the particulate matter was removed by centrifugation. Nonidet P-40 (1% final concentration) was added to the supernatant and the extract was loaded onto a 0.25-ml DEAE-Sepharose column. The column was washed with 1.5 ml of each of the following: lysis buffer plus 1% Nonidet P-40; lysis buffer plus 0.25 M NaCl; lysis buffer plus 0.5 M NaCl. The eluant was collected in 0.5-ml fractions and CT activity was located in the 0.25 M NaCl wash. CT that was eluted from the column was completely inactive in the absence of lipid activators.

CT Assay

The delipidated CT preparation (2 µg) was assayed for activity in a buffer composed of lipid activator (80 µM PtdCho, 80 µM oleic acid, 1:1), 2 mM CTP, 20 mM MgCl(2), 125 mM bis-Tris-HCl, pH 7.0, 1 mM phospho[^14C]choline (specific activity 0.2 Ci/mmol), in a final volume of 40 µl(21) . The reaction mixture was incubated at 37 °C for 10 min and the reaction was terminated by the addition of 5 µl of 0.5 M EDTA, pH 8.0, and the tubes were vortexed and placed on ice. Next, 30 µl of the supernatant was spotted on Silica Gel G layers that were developed in methanol, 0.6% NaCl, ammonium hydroxide (10:10:1, v/v). CDP-[^14C]choline was identified by co-migration with a standard scraped from the plate and quantitated by liquid scintillation counting.

CPT Assay

BAC1.2F5 microsomes were prepared as described previously (51) and were used to assay CPT activity as described by Hjelmstad and Bell(52) . Briefly, microsomes were incubated with Triton X-100/diolein/egg PtdCho mixed micelles (in 80 mM MgCl(2), 200 mM MOPS-NaOH, pH 7.5) for 5 min. The reaction was started by the addition of CDP-[^14C]choline (0.5 mM; specific activity 4 mCi/mmol) and incubated at 25 °C for 10 min. The reaction was stopped by transferring samples to Whatmann 3MM filter disks which were washed twice with 5% trichloroacetic acid and once with 1% trichloroacetic acid to remove unincorporated CDP-[^14C]choline. The amount of [^14C]PtdCho formed was quantitated by liquid scintillation counting.

Choline Kinase Assay

BAC1.2F5 cells were lysed in buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 0.25 M sucrose, 1% aprotinin, 1 mM EDTA, 1 mM NaF) on ice for 30 min followed by sonication. Choline kinase activity was measured essentially as described by Pelech et al.(53) in an incubation containing 100 mM Tris-HCl, pH 8.5, 100 mM MgCl(2), 2 mM [^14C]choline chloride (specific activity 1.5 mCi/mmol), 10 mM ATP, and BAC1.2F5 cell lysate (0-300 µg of protein) in a final volume of 100 µl. The reaction mixture was incubated at 37 °C for 30 min and the reaction was stopped by placing the tubes in a boiling water bath for 2 min. Precipitated protein was removed by centrifugation and phospho[^14C]choline was separated from [^14C]choline by thin layer chromatography on Silica Gel G layers developed with methanol, 0.6% NaCl, 28% ammonium hydroxide (10:10:1, v/v). The areas corresponding to choline and phosphocholine (determined by co-migration with standards) were scraped and the radioactivity was measured by liquid scintillation counting. Choline kinase activity was linear with protein concentration.

Metabolic Labeling

BAC1.2F5 cells were grown in complete medium for 48 h. The medium was replaced with choline-free Dulbecco's modified Eagle's medium plus 15% dialyzed fetal calf serum and 25% dialyzed L-cell conditioned medium and 24 h later, fresh complete choline-free medium was added. In preliminary experiments, cells were prelabeled while growing for 4 days in 2 µM [^3H]choline (3 Ci/mmol). Growth rates were comparable to cells incubated in complete medium during the prelabeling period and also during subsequent starvation for choline. Cells were then transferred to choline-free medium for different time periods and the levels of intracellular choline metabolites were measured with time. The intracellular water soluble choline-derived pools dropped 66% by 6 h, and as the starvation period was extended the soluble pool gradually decreased 72% by 24 h compared to the pool size at the initiation of the time course. The major metabolite remaining at the end of 24 h was phosphocholine.

For the experiments reported in this paper, either LPC or ET-18-OCH(3) was added followed by [^3H]choline (2 µM, 1.5 µCi/ml final concentrations) 5 min later. After the intervals indicated, the adherent cells were washed with ice-cold PBS and harvested by scraping into 1 ml of PBS. The cells were pelleted by centrifugation. The soluble and organic choline-derived metabolites were separated by extraction using the method of Bligh and Dyer(54) , and the amount of choline incorporated into each fraction was determined by scintillation counting. The distribution of label among the phospholipid classes was determined by thin-layer chromatography on Silica Gel G layers developed in chloroform/methanol/acetic acid/water (50:25:8:4, v/v) and the distribution of label among the soluble choline-derived metabolites was determined by chromatography on Silica Gel G thin layers developed with methanol, 0.6% NaCl, 28% ammonium hydroxide (10:10:1, v/v).

LPC uptake into BAC1.2F5 cells was measured by labeling 2 times 10^6 cells/60-mm dish with 50 µM egg LPC plus [1-^14C]palmitoyl-LPC (0.15 mCi/ml). At 1, 2, 4, and 6 h the cells were washed twice with PBS and the lipids extracted(54) . The lipid phase was counted and fractionated by thin-layer chromatography on Silica Gel G layers developed with chloroform/methanol/acetic acid/water (50:25:8:3, v/v) and the distribution of label between LPC, PtdCho, and fatty acid/neutral lipid was determined.

Digitonin Fractionation

BAC1.2F5 cells were fractionated into soluble and particulate fractions using the digitonin permeabilization procedure(11, 13, 55) . Briefly, cells were either untreated controls or treated with 12 µM ET-18-OCH(3) for 5 h, scraped from the dish and gently resuspended in digitonin buffer (0.4 mg/ml digitonin, 1 mM phenylmethylsulfonyl fluoride, aprotinin (0.16 trypsin inhibitor units/ml), 50 mM sodium fluoride, 100 µM sodium orthovanadate, 100 nM okadaic acid, 3 mM EDTA, 0.25 M sucrose, 10 mM Tris-HCl, pH 7.4). The cells were centrifuged for 10 min at 1800 times g. Aliquots of the supernatant (cytosol) or pellet (particulate) were analyzed for protein content, lactate dehydrogenase activity, CT activity, and immunoprecipitated and immunoblotted to determine CT protein levels. Lactate dehydrogenase was used as a marker for soluble proteins and its activity was determined using a colorimetric assay kit purchased from Sigma. Immunoprecipitation and immunoblotting were performed as described previously(21) . Protein was determined by the Bradford method(56) .

LPC Mass Determination

Cells were incubated in medium supplemented with 200 µM LPC for 1 h, washed twice with ice-cold PBS, and the lipids extracted(54) . LPC was separated from total lipids by thin-layer chromatography on Silica Gel H layers developed with chloroform/methanol/acetic acid/water (50:25:8:3, v/v). The bands corresponding to LPC (determined by comigration with standards) and PtdCho were scraped from the thin-layer and the amount of LPC and PtdCho were determined by measuring the phosphorus content of the bands as described(57) .


RESULTS

LPC and ET-18-OCH(3) Inhibit Choline Incorporation into Phospholipids

The incorporation of [^3H]choline into phospholipid was measured in BAC1.2F5 cells incubated with LPC to determine the effect of LPC on PtdCho synthesis via the CDP-choline pathway. LPC treatment significantly reduced [^3H]choline incorporation into phospholipid (Fig. 2, Panel A). A 50% reduction in the radiolabeling of the PtdCho pool occurred at 12.5 µM LPC and inhibition approached 90% at the highest LPC concentration tested (200 µM). The highest LPC concentration did not cause a significant release of cellular lactate dehydrogenase and, together with the data below indicating the accumulation of soluble PtdCho precursors, showed that LPC effects could not be attributed to nonspecific permeabilization of cell membranes. We radiolabeled cells with 50 µM [^14C]LPC for 1 h and found the label primarily in PtdCho (71.1%) and neutral lipid (20.3%) with the remaining 8.6% in LPC indicating that exogenous LPC was rapidly converted to PtdCho by BAC1.2F5 cells. The steady state level of LPC in BAC1.2F5 cells was measured by equilibrium labeling of the phospholipid pools with [P]orthophosphate as described(21) . These data showed that LPC comprised 2.5 ± 1.2% of the total phospholipid pool. Cellular LPC levels were also determined by lipid phosphorus measurements. In control cells, LPC levels were 7.9 ± 0.95 nmol/10^7 cells compared to PtdCho which was 209 ± 3.7 nmol/10^7 cells. Following the exposure of BAC1.2F5 cells to 200 µM LPC for 1 h, the PtdCho content was essentially unchanged (203 ± 7.7 nmol/10^7 cells), whereas the LPC level increased 3-fold to 24.6 ± 4.2 nmol/10^7 cells. ET-18-OCH(3) was a more potent inhibitor of PtdCho synthesis than LPC (Fig. 2, Panel B). A 50% reduction in [^3H]choline incorporation into PtdCho occurred between 1 and 2 µM ET-18-OCH(3). ET-18-OCH(3) at 12 µM did not cause significant release of lactate dehydrogenase during the time course of the experiments. These data suggested that ET-18-OCH(3) interfered with PtdCho synthesis by acting as an LPC analog.


Figure 2: LPC inhibited [^3H]choline incorporation into phospholipid. BAC1.2F5 cells were grown for 2 days to a density of 2.8 times 10^6 cells/60 mm-dish. The cells were washed with PBS and incubated in choline-free medium for 18 h. The medium was then replaced with fresh choline-free medium, and 1 h later, the indicated concentrations of LPC (Panel A) or ET-18-OCH(3) (Panel B) were added. Five min later, [^3H]choline (1.5 µCi/ml, 2 µM final concentration) was added and the cells were incubated for an additional 4 h. Cells were washed and harvested by scraping into ice-cold PBS, and the amounts of [^3H]choline incorporated into the phospholipid fractions were determined by scintillation counting as described under ``Experimental Procedures.''



We investigated the metabolic point of inhibition of the PtdCho biosynthetic pathway by radiolabeling cells with [^3H]choline for a 6-h period in the presence and absence of 200 µM LPC (Fig. 3). The soluble choline-derived pool was depleted prior to the addition of radiolabel of known specific activity (0.75 mCi/µmol) to ensure that the analysis of [^3H]choline-derived metabolites reflected the actual distribution of the PtdCho precursors. LPC had a small inhibitory effect on the total [^3H]choline uptake into the cells (Fig. 3, Panel A), thus indicating that the concentrations of LPC used neither increased the nonspecific permeability of cells nor blocked the transport of choline across the cell membrane. Since the soluble choline metabolite pool must be labeled prior to incorporation of choline into PtdCho, the appearance of label in PtdCho always began after about a 1-h lag period. However, the inhibition of PtdCho synthesis by LPC was clearly evident by 2 h of incubation (Fig. 3, Panel B). The soluble pool containing all three of the choline-derived precursors of PtdCho attained metabolic equilibrium in untreated cells within 2 h and was maintained at this level due to the flux through the pathway (Fig. 3, Panel C). On the other hand, the soluble precursor pool continued to expand in cells treated with LPC indicating that inhibition of the pathway occurred prior to the CPT step (Fig. 1) and resulted in the accumulation of one or more of the three soluble precursors: choline, phosphocholine, or CDP-choline. LPC also inhibited choline incorporation into PtdCho in cells not deprived of choline, consistent with the observations of others(22, 23, 58) .


Figure 3: Time course for [^3H]choline uptake by BAC1.2F5 cells in the presence and absence of LPC. Cells were grown to a density of 6.5 times 10^6 cells/100-mm dish and the intracellular choline pools were reduced by incubation with choline-free medium as described in the legend to Fig. 2. At time 0, 200 µM LPC was added, and 5 min later, [^3H]choline (1.5 µCi/ml, 2 µM final concentration) was added and the cells were incubated for the indicated times, and harvested. An aliquot was removed to determine the total [^3H]choline incorporated into the cells (Panel A). The cell pellets were extracted to determine the amounts of [^3H]choline incorporated into the phospholipid (Panel B) or soluble (Panel C) fractions as described under ``Experimental Procedures.'' Untreated cells (bullet); LPC-treated cells (circle). The data points are the means of duplicate samples from a representative experiment.



The soluble fraction from the experiment described in Fig. 3was analyzed by thin-layer chromatography to determine the distribution of label among the soluble [^3H]choline-derived metabolites (Fig. 4). The uptake of choline within the first hour was the same for both control and treated cells, whereas choline continued to accumulate with time after LPC addition (Fig. 4, Panel A) indicating that the restriction point in the pathway was downstream of choline transport. Likewise, phosphocholine continued to accumulate in LPC-treated cells to a level significantly higher than in control cells (Fig. 4, Panel B) and LPC did not inhibit choline kinase activity in vitro indicating that this enzyme was not a LPC target. In contrast, the production of CDP-choline was dramatically reduced in LPC-treated cells (Fig. 4, Panel C), indicating that LPC blocked the PtdCho biosynthetic pathway at the CT step (Fig. 1).


Figure 4: LPC inhibited the formation of CDP-choline in vivo. The soluble fractions from the experiment described in Fig. 3(Panel C) were fractionated by thin-layer chromatography as described under ``Experimental Procedures'' to determine the distribution of label among the soluble choline-derived metabolites. Panel A, choline (Cho); Panel B, phosphocholine (Pcho); and Panel C, CDP-choline (CDP-Cho). Untreated cells (bullet); LPC-treated cells (circle).



An identical series of experiments and data analysis was carried out with BAC1.2F5 cells treated with 12 µM ET-18-OCH(3). In both cases, PtdCho synthesis was effectively blocked. Choline and phosphocholine accumulated in ET-18-OCH(3)-treated cells up to 2 h, but then decreased at the 4- and 6-h time points. ET-18-OCH(3) also blocked de novo PtdCho synthesis in cells not deprived of choline as reported by others(37, 38, 39, 40, 41, 42, 43) . This difference between ET-18-OCH(3)-treated and LPC-treated cells was attributed to the cytotoxicity of ET-18-OCH(3) since the viable cell number decreased in the cultures at the 4- and 6-h time points. This finding was consistent with the rapid cytotoxic action of ET-18-OCH(3)(25, 26, 27, 28, 29, 30, 31, 32, 33) and other antineoplastic phospholipids (41, 42, 43) observed in other cell systems.

The specificity of the inhibition of PtdCho synthesis with respect to the polar headgroup on the lysophospholipid was addressed by comparing the effects of LPC, LPE, and LPS on the incorporation of [^3H]choline into BAC1.2F5 cells (Fig. 5). LPS and LPE had no significant effect on the amount of [^3H]choline incorporated into the phospholipid pool of BAC1.2F5 cells. Thus, the inhibition of PtdCho synthesis was specific for lysophospholipids with the choline headgroup.


Figure 5: LPE and LPS did not inhibit PtdCho synthesis. BAC1.2F5 cells were grown to a density of 2.8 times 10^6 cells/60-mm dish and the intracellular choline pools were reduced by incubation with choline-free medium as described in the legend to Fig. 2. Cells were then treated with 100 µM of the indicated lysophospholipid, and 5 min later, [^3H]choline (1.5 µCi/ml, 2 µM final concentration) was added and the cells were incubated for 4 h. The extent of [^3H]choline incorporation into the phospholipid pool was determined as described under ``Experimental Procedures.'' The values are means ± range of duplicate samples.



LPC did not block PtdCho synthesis by acting as an inhibitor of cell growth. BAC1.2F5 cells were synchronized in G(1) by the transient removal of CSF-1 (49) and then growth factor was added along with [^3H]thymidine and increasing concentrations of LPC (0-200 µM). After 24 h of incubation, the maximum decrease in [^3H]thymidine incorporation was 11% at 200 µM LPC and correlated with 10% fewer cells after the first round of cell division compared to control, untreated cultures. Lower concentrations of LPC had no effect and after 24 h, the cultures treated with 200 µM LPC recovered normal doubling times. These data indicated that the metabolic effects of LPC could not be attributed to toxicity. In contrast, 12 µM ET-18-OCH(3) blocked [^3H]thymidine incorporation into BAC1.2F5 cells (>90%) and the number of viable cells was reduced to 8% of the starting cell density after 24 h. These findings with the ether-linked LPC analog were expected and are consistent with its cytotoxic effects in many cell types.

Effect of LPC and ET-18-OCH(3) on CT in Vivo

The inhibition of CT activity by LPC and ET-18-OCH(3) could be due to alterations in the amount of CT protein, its phosphorylation state, or its degree of membrane association. BAC1.2F5 cells contain 3 phosphorylated CT isoforms that are distinguished by their electrophoretic mobilities, and the more slowly migrating, hyperphosphorylated forms are less active than the hypophosphorylated form(21) . To determine whether LPC or ET-18-OCH(3) reduced CT activity by either altering the distribution of electrophoretic forms or the amount of enzyme per cell, BAC1.2F5 cells were incubated with either 100 µM LPC or 12 µM ET-18-OCH(3) for 4 h, and the amount and type of CT present was determined by immunoprecipitation and immunoblotting (Fig. 6). There was no difference between the amount or the distribution of CT forms in either LPC- or ET-18-OCH(3)-treated cells.


Figure 6: Neither LPC nor ET-18-OCH(3) altered the cellular content of CT. BAC1.2F5 cells were grown to a density of 9 times 10^6 cells/100-mm dish and treated with either 100 µM LPC or 12 µM ET-18-OCH(3) for the indicated times. The cells were then harvested, lysed, and immunoprecipitated with CT antibody. The immunoprecipitates were fractionated by gel electrophoresis and the content and distribution of CT isoforms was determined by immunoblotting as described under ``Experimental Procedures.''



We determined the effect of LPC and ET-18-OCH(3) on the binding of CT to membranes using the digitonin release assay (Fig. 7). In untreated control BAC1.2F5 cells, CT protein was found primarily in the soluble fraction released by 0.4 mg/ml digitonin along with >96% of the total lactate dehydrogenase. The localization, as determined by immunoblotting (Fig. 4), was corroborated by enzyme assays in the presence of the maximum concentration of lipid activator which showed >90% of the total CT activity was recovered in the soluble fraction and <10% in the particulate fraction. Treatment of the cells with 200 µM LPC for 4 h did not change the distribution of CT (Fig. 7, Panel A). In contrast, treatment of the cells with 12 µM ET-18-OCH(3) for 4 h resulted in the translocation of CT from the digitonin-soluble fraction to the digitonin-insoluble or particulate fraction (Fig. 7, Panel B). CT translocation to the particulate fraction induced by ET-18-OCH(3) was also evident from CT activity assays which showed 45% of the total CT activity was associated with the digitonin-insoluble fraction in ET-18-OCH(3)-treated cells.


Figure 7: Effect of LPC and ET-18-OCH(3) on cellular distribution of CT. Exponentially growing BAC1.2F5 cells were treated with either 200 µM LPC (Panel A) or 12 µM ET-18-OCH(3) (Panel B) for 4 h. Treated and control cells were then separated into particulate and soluble fractions by incubation with digitonin as described under ``Experimental Procedures.'' The distribution of CT between the soluble and particulate fractions was determined by measuring the levels of CT enzyme activity (Bar Graphs) and the levels of CT protein were determined by immunoprecipitation and immunoblotting (Gel Inserts). The total CT activities (pmol/min/dish) were: Panel A, control, 280.4 ± 28.8; LPC-treated, 307.5 ± 32.2; Panel B, control, 281.0 ± 21.5; ET-18-OCH(3)-treated, 228.6 ± 12.



LPC and ET-18-OCH(3) Inhibited CT Activity in Vitro

To corroborate the conclusions from the in vivo experiments that CT was a target for LPC and ET-18-OCH(3), the effect of these agents on CT activity in vitro was determined (Fig. 8). Using BAC1.2F5 cell extracts, we were able to demonstrate the inhibition of CT activity by either LPC or ET-18-OCH(3) (not shown). The BAC1.2F5 system was not ideal for the detailed analysis of the effects of lysophospholipids on CT activity because CT was a minor protein component and the crude extracts contained unknown quantities of lipid activators. Therefore, we used a baculovirus expression system to obtain large amounts of recombinant CT protein for enzymatic analysis(50) . Recombinant rat CT expressed in Sf9 cells was bound to a DEAE-cellulose column equilibrated with the cell lysis buffer, and the column was then washed with 5 column volumes of 1% Nonidet P-40 in the same buffer to remove endogenous lipid activators. The detergent elution step removed the endogenous lipids in the CT sample. The column was next washed with buffer to eliminate the detergent and the CT was subsequently eluted with 0.25 M NaCl. This procedure was similar to the methods used by Cornell (8) and Luche et al.(50) to remove endogenous lipid activators bound to CT. CT purified in this manner was completely devoid of enzymatic activity in the absence of lipid activators (not shown, see (8) and (50) ), thus allowing complete control over the lipid composition of the assay. PtdCho:oleic acid (1:1) vesicles were used as lipid activator and the apparent K(m) for this activator mixture was 10 µM. LPC inhibition of CT activity was detected at 5 µM, reached 50% at 25 µM, and reached 80% at 80 µM LPC in the presence of 80 µM PtdCho:oleic acid vesicles (Fig. 8, Panel A). ET-18-OCH(3) was also a potent inhibitor of purified CT with 50% inhibition of the CT reaction occurring at 8 µM ET-18-OCH(3) (Fig. 8, Panel C). Experiments with LPC and ET-18-OCH(3) indicated that the inhibition was competitive with respect to the lipid activator (Fig. 8, Panels B and D). LPS and LPE did not inhibit CT activity. ET-18-OCH(3) or LPC inhibition was noncompetitive with respect to the CT substrates, CTP and phosphocholine (data not shown). These data verify that LPC and ET-18-OCH(3) were inhibitors of CT activity and support the hypothesis that these lysophospholipids acted by interfering with lipid activation.


Figure 8: LPC and ET-18-OCH(3) inhibited CT activity in vitro. CT was expressed in insect cells using a recombinant baculovirus and endogenous lipids were removed by ion-exchange chromatography as described under ``Experimental Procedures.'' CT activity was determined in the presence of 80 µM PtdCho:oleic acid (1:1) vesicles as the lipid activator. Panel A, concentration dependence for the inhibition of CT activity by LPC. Panel B, kinetic analysis of CT inhibition by LPC. Panel C, concentration dependence for the inhibition of CT activity by ET-18-OCH(3). Panel D, kinetic analysis of CT inhibition by ET-18-OCH(3). The dependence of CT activity on the concentration of the lipid activator either in the absence or presence of two different LPC or ET-18-OCH(3) concentrations was analyzed using double-reciprocal plots. Data points are the average of duplicate reactions from a representative experiment.



LPC Did Not Inhibit CPT or Choline Kinase

Although these data show that LPC inhibits CT, we also investigated the possibility that LPC inhibited CPT, the membrane-bound enzyme that utilizes CDP-choline plus diacylglycerol to form PtdCho. A mammalian CPT has not been cloned, and therefore, studies were limited to activity assays in cell homogenates. Microsomes isolated from BAC1.2F5 cells were incubated with LPC and CPT activity was measured. There was no significant difference between CPT activity in control microsomes (3 nmol/min/mg protein) and microsomes assayed in the presence of 10-200 µM LPC. CPT was also assayed in microsomes prepared from BAC1.2F5 cells in the absence or presence of ET-18-OCH(3) at concentrations up to 80 µM. We did not observe inhibition of CPT activity under these assay conditions. These data indicated that CPT was not a target for ET-18-OCH(3); however, this conclusion should be corroborated with kinetic experiments using homogeneous CPT when methods to isolate and reconstitute this protein becomes available. One interpretation of the data showing the effect of LPC on the distribution of label in choline metabolites in the in vivo labeling experiment (Fig. 4) would be that LPC inhibited choline kinase in addition to CT. To determine if choline kinase may be effected by LPC, choline kinase was assayed in BAC1.2F5 lysates in the presence and absence of LPC. There was no significant difference between choline kinase activity in the presence or absence of LPC (1.4 ± 0.26 nmol/min/mg of protein in control incubations and 1.8 ± 0.32 in incubations containing 60 µM LPC, n = 3).


DISCUSSION

Our results lead to the conclusion that LPC acts as a regulator of the CDP-choline pathway for PtdCho synthesis by inhibiting the CT step as outlined in Fig. 1. The acylation of LPC to PtdCho circumvents the de novo biosynthetic pathway and provides PtdCho for cell growth, thereby providing a compelling physiological rationale for the ability of LPC to act as a negative regulator of CT activity. Negative regulation of the CDP-choline pathway is specific for LPC, since lysophospholipids lacking the choline headgroup do not inhibit PtdCho synthesis (Fig. 5). CT is the target for LPC based on the accumulation of choline and phosphocholine accompanied by a decrease in CDP-choline in LPC-treated cells (Fig. 3) coupled with the inhibition of CT activity by lysophospholipids in vitro (Fig. 8). An alternate explanation for our results is that LPC treatment increases the cellular content of PtdCho, which in turn regulates CT activity, and therefore, it is important to address whether LPC directly inhibits CT activity in vivo. Vance and co-workers(17, 22, 23, 53) established that a correlation exists between the cellular PtdCho content and flux through the CDP-choline pathway. They concluded that the mechanistic basis for this correlation is that increased membrane PtdCho content causes a decrease in the membrane-associated (``active'') CT. In our experiments the digitonin fractionation assay did not reveal a clear difference between the distribution of CT between soluble and particulate fractions in control and LPC-treated cells (Fig. 7). Greater than 90% of the total immunoreactive CT protein and CT activity as well was found in the soluble fraction in both cases, although control cells were rapidly synthesizing PtdCho via the CDP-choline pathway and the LPC-treated cells were not. Therefore, a change in the ratio of soluble and particulate forms of CT does not explain the LPC effects on de novo PtdCho synthesis. The most compelling evidence that LPC directly affects CT activity is the inhibition of PtdCho biosynthesis and CT activity by the non-metabolizable LPC analog, ET-18-OCH(3) ( Fig. 2and Fig. 8). ET-18-OCH(3) is not converted to PtdCho, and is a more potent inhibitor of PtdCho synthesis than LPC, suggesting that the metabolic conversion of LPC to PtdCho actually diminishes its effect on CT and contributes significantly to the higher dose of LPC compared to ET-18-OCH(3) required to inhibit the CDP-choline pathway together with the lower affinity of CT for LPC determined in vitro (Fig. 8). Metabolic labeling experiments show that LPC is rapidly converted to PtdCho by BAC1.2F5 cells resulting in only a 3-fold increase in the LPC content of cells treated with 200 µM LPC for 1 h. PtdCho and LPC exist in a dynamic equilibrium regulated by the activities of phospholipases and acyltransferases/transacylases, and thus higher concentrations of PtdCho may reflect higher levels of LPC, which in turn, may be the actual regulator of CT activity. Our finding that lysophospholipids are potent inhibitors of the CDP-choline pathway indicate that LPC levels should be measured in experiments correlating phospholipid content with PtdCho synthesis. Our data also indicate that stimulation of PLA(2) activity, which results in LPC production as well as the release of arachidonate or other fatty acids, may play a role in the control of the CDP-choline pathway by increasing the LPC pool.

Our experiments are consistent with the idea that LPC and ET-18-OCH(3) bind to the lipid activation domain of CT, but do not induce the conformational changes required to activate the enzyme. Competitive inhibition of CT by ET-18-OCH(3) with respect to PtdCho:oleic acid lipid activator (Fig. 8) and noncompetitive inhibition with respect to phosphocholine and CTP point to the lysophospholipids binding to the same site as the lipid activators. CT is known to absolutely require lipid activators for catalytic activity(7, 8) , and the use of the non-metabolizable LPC analog allows the clear delineation of lysophospholipid interactions with CT in vivo and in vitro. CT is extracted into the soluble fraction in control and LPC-treated cells, whereas significant quantities are localized in the particulate fraction in ET-18-OCH(3)-treated cells (Fig. 7). ET-18-OCH(3) is known to partition into biological membranes (59) and the association of CT with the particulate fraction of ET-18-OCH(3)-treated cells is consistent that the idea that ET-18-OCH(3) binds to, but does not activate CT. Active lysophospholipases and transacylases that metabolize LPC during the preparation of the cells for the cellular fractionation experiments may explain the difference in the distribution of CT between soluble and particulate fraction in LPC- and ET-18-OCH(3)-treated cells. Our data are not consistent with the report of Tronchere et al.(40) who concluded that ET-18-OCH(3) releases CT from the membrane. The reason for the difference between the two conclusions is not clear, but the experimental approaches were quite different. Tronchere et al.(40) added ET-18-OCH(3) to a crude cell lysate and then separated membrane and soluble fractions by centrifugation, whereas we determined the degree of membrane association following the treatment of intact cells with ET-18-OCH(3). Thus, it remains possible that CT is localized in the particulate fraction of ET-18-OCH(3)-treated cells due to alterations in membrane composition resulting from the inhibition of PtdCho synthesis. The high percentage of CT associated with the particulate fraction coupled with the block in PtdCho synthesis in ET-18-OCH(3)-treated cells illustrates that increased CT membrane-association determined by the digitonin release assay and increased rates of PtdCho synthesis do not necessarily correlate.

Since CT activity is necessary for cell survival(3, 4) , it is clear from our work that CT is a relevant intracellular target for antineoplastic phospholipids typified by ET-18-OCH(3). Antineoplastic phospholipids are known to inhibit the incorporation of choline into PtdCho and reduce cellular PtdCho levels(36, 37, 38, 39, 40) . Hexadecylphosphocholine, another antineoplastic LPC analog, also partially inhibits CT activity as assayed in crude cell homogenates (44) . Our findings define the mechanism for LPC and ET-18-OCH(3) inhibition of CT as competitive with respect to lipid activators, suggesting that the biological effects of ET-18-OCH(3) and other antineoplastic phospholipids may be suppressed by the addition of either LPC to circumvent the CDP-choline pathway or by oleic acid, a potent lipid activator of CT that may compete with ET-18-OCH(3) for CT binding and antagonize the effects of the drug. Defining the contribution of blocking PtdCho synthesis to the cytotoxicity of antineoplastic phospholipids will be an important step in understanding the mechanism of action of these drugs.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM 45737 (to S. J.), Cancer Center (CORE) Support Grant CA 21765, and the American Lebanese Syrian Associated Charities. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38101-3018. Tel.: 901-522-0494; Fax: 901-525-8025.

(^1)
The abbreviations used are: PtdCho, phosphatidylcholine; CSF-1, colony-stimulating factor 1; ET-18-OCH(3), 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; CT, CTP:phosphocholine cytidylyltransferase; CPT, choline phosphotransferase; LPC, 1-acyl-sn-glycero-3-phosphocholine; LPS, lysophosphatidylserine; LPE, lysophosphatidylethanolamine; PBS, phosphate-buffered saline; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,2-diol; MOPS, 4-morpholinepropanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Robyn Roberts, Huong Nguyen, and Margarita Perez Pecha for their expert technical assistance.


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