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INTRODUCTION |
Apoptosis, or programmed cell death, is a means by which organisms
rid themselves of unwanted cells without the induction of an
inflammatory response (1, 2). Apoptosis occurs naturally during the
development process and for the removal of damaged cells or normal
cells that have reached the end of their life span. Misregulation of
the apoptotic process is believed to be a major means by which cells
can become cancerous (3, 4), and conversely many chemotherapeutic drugs
for the treatment of cancer do so by preferential induction of
apoptosis in cancer cells (5, 6). In a recent study, 31P
NMR spectroscopy was used to study alterations in cellular metabolites upon treatment with a variety of apoptosis inducing drugs as a means of
identifying a marker associated with the induction of apoptosis (7).
All of the agents tested resulted in an increase in the level of
CDP-choline suggesting an inhibition of phosphatidylcholine (PC)1 synthesis at the
cholinephosphotransferase step (8, 9).
PC is the most abundant lipid present in eukaryotic cell membranes
comprising ~50% of cellular phospholipid mass (10). PC is
synthesized almost exclusively via the three step Kennedy
pathway in most eukaryotic cells types (11-14). In this pathway,
choline is phosphorylated by choline kinase to produce phosphocholine which is subsequently converted to CDP-choline by CTP:phosphocholine cytidylyltransferase with the final step catalyzed by
cholinephosphotransferase through the transfer of phosphocholine from
CDP-choline to diacylglycerol to produce an intact PC molecule. Genetic
inactivation of PC synthesis in Chinese hamster ovary (CHO) cells by
shifting a cell line containing a temperature-sensitive allele of
CTP:phosphocholine cytidylyltransferase to the non-permissive
temperature resulted in cell death by apoptosis, and this was prevented
by expression of a wild type CTP:phosphocholine cytidylyltransferase
cDNA (15).
One of the compounds used in the search for alterations in cellular
metabolites using 31P NMR spectroscopy during the induction
of apoptosis was farnesol, a catabolite of the isoprenoid/cholesterol
biosynthetic pathway (16). Farnesol is especially interesting in that
it has been demonstrated to preferentially induce apoptosis in several
transformed cells versus untransformed cells (17, 18).
Metabolic analyses using labeled choline determined that the addition
of farnesol to several cell types in culture resulted in a rapid and
dramatic inhibition of PC synthesis, and the metabolic block was due to inhibition at the cholinephosphotransferase step (8, 9, 19).
Farnesol-induced apoptosis could be specifically rescued by exogenous
PC or diacylglycerol administration, whereas the addition of other
phospholipids was ineffective (8, 20). An in vitro
enzymatic analysis of cholinephosphotransferase activity present in
cell membranes indicated that farnesol inhibited PC synthesis by
directly competing with diacylglycerol for binding to the active site
of cholinephosphotransferase (9). Thus, it was concluded that farnesol
directly competed with diacylglycerol for binding to the
cholinephosphotransferase active site resulting in a dramatic
inhibition of PC synthesis that directly led to apoptosis (9).
Our recent isolation of the first mammalian cholinephosphotransferase
cDNA (21, 22) has allowed us to more precisely determine how
farnesol induces apoptosis. Our major findings include the observation
that over-expression of human cholinephosphotransferase in CHO cells
prevented farnesol-induced inhibition of PC synthesis; however, it did
not prevent farnesol-induced apoptosis. In addition, exogenous
administration of diacylglycerol significantly reduced farnesol-induced
apoptosis but did not rescue PC synthesis. Thus we have uncoupled the
inhibition of PC synthesis by farnesol from the ability of farnesol to
induce apoptosis. To further this conclusion, a mixed micelle assay was
developed for the determination of cholinephosphotransferase activity,
and it did not reveal significant inhibition of the enzyme by farnesol
or its phosphorylated derivatives. Farnesol-induced apoptosis is not
via inhibition of PC synthesis through direct competition
with diacylglycerol for the cholinephosphotransferase active site, but
is instead likely through a diacylglycerol-directed mechanism that is
downstream of PC synthesis.
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EXPERIMENTAL PROCEDURES |
Materials--
[methyl-14C]CDP-choline,
[methyl-14C]choline, and
[1,2-14C]ethanolamine were purchased from American
Radiolabeled Chemicals. Custom oligonucleotides, T4 DNA ligase,
Taq polymerase, and all tissue culture products were
obtained from Life Technologies, Inc. Lipids were purchased from Avanti
Polar Lipids or Sigma. The pTRE2 vector, pTK-Hyg vector, and the Tet-On
CHO-K1 cell line were purchased from CLONTECH. The
T7 monoclonal antibody coupled to horseradish peroxidase was a product
of Novagen. Poly(ADP-ribose) polymerase (PARP) antibodies were
purchased from PharMingen. Secondary antibodies were from Bio-Rad. All
other reagents were of the highest quality commercially available.
Cell Line Construction--
Tet-On CHO-K1 cells were
co-transfected with 10 µg the pTRE2 vector containing a T7
epitope-tagged version of CEPT1 (21) and 1 µg of the pTK-Hyg
selection vector using the calcium chloride method (23) and
subsequently incubated in Dulbecco's modified Eagle's media
containing 5% fetal bovine serum in a humidified atmosphere containing
5% CO2. After 24 h, media was changed and 300 µg/ml
G418 and 400 µg/ml hygromycin were added. The antibiotic-containing media were changed every 48 h for 3 weeks to select for stable integrants. Single colonies were isolated, and cell lines were tested for inducible expression of CEPT1 by the addition of 2 µg/ml
of doxycycline for 24 h. Cellular membranes were isolated and
incubated with SDS-polyacrylamide gel electrophoresis sample buffer at 37 °C for 20 min, separated on a 10% SDS-polyacrylamide gel electrophoresis gel, and transferred to polyvinylidene difluoride membranes. Blots were probed with a T7 epitope tag monoclonal antibody
(1:10,000) coupled to horseradish peroxidase for subsequent detection
using the ECL (Amersham Pharmacia Biotech) system. Confirmation of
CEPT1-inducible expression was determined by performing
cholinephosphotransferase enzyme assays on isolated cellular membrane
protein preparations.
Enzyme Assays--
CHO-K1 cells induced to overexpress CEPT1
(CHO-AA8-CEPT1) were placed on ice and washed twice with ice-cold
phosphate-buffered saline, scraped into a microfuge and centrifuged at
15,000 × g for 5 min at 4 °C. The cell pellet was
resuspended in 0.5 ml of 10 mM Hepes-HCl (pH 7.4), 50 mM KCl, 1 mM EDTA, and complete protease inhibitor mixture (Roche Molecular Biochemicals) and passed through a
23-gauge needle 20 times to lyse the cells. The mixture was centrifuged
at 15,000 × g for 30 s at 4 °C to pellet
unbroken cells and nuclei. The supernatant was centrifuged at
450,000 × g for 15 min at 4 °C to pellet cellular
membranes. Membranes were resuspended in 10 mM Hepes-HCl
(pH 7.4), 50 mM KCl, 1 mM EDTA, and complete
protease inhibitor mixture (Roche Molecular Biochemicals) by using a
Teflon pestle and stored at
70 °C. Two methods were used to assess
cholinephosphotransferase activity. The first disperses the
diacylglycerol into solution without the aid of solubilizing detergents
(9) and was performed in an assay buffer containing 175 mM
Tris-EDTA (pH 8.0), 8 mM MgCl2, 0.5 mM EDTA, 1 mg/ml bovine serum albumin, 2 mM
di18:1 diacylglycerol, and 15 µg of microsomal protein. Some assays
contained farnesol delivered in ethanol (or ethanol only control) and
were preincubated for 0-30 min at 37 °C (9). Radiolabeled
[14C]CDP-choline (0.1 mM, 2,000 dpm/nmol) was
added to initiate the reaction and tubes were incubated at 37 °C for
10 min. Assays were terminated by the addition of chloroform/methanol
(2/1, v/v) and 0.9% KCl to facilitate phase separation. The lower
organic phase was washed twice with an equal volume of 40% methanol
(v/v), and the PC product in the organic phase was dried in
scintillation vials and radiolabel was determined. The second method
was a sodium cholate-mixed micelle method performed in 50 mM Tris-HCl (pH 8.0), 20 mM MgCl2,
20% glycerol, with 10 mol% diacylglycerol and 10 mol% PC in 1%
sodium cholate using 10-25 µg of membrane protein as the enzyme
source. The assay mix was incubated at 25 °C for 5 min to allow for
mixed micelle formation, then initiated by the addition of CDP-choline
(0.4 mM, 2000 dpm/nmol), and incubated at 25 °C for 20 min. Cholinephosphotransferase activity determined by using the mixed
micelle assay increased linearly for both substrates. The assay
contained at least a 2-fold greater than Km concentration of each substrate. Assays were terminated, and product was isolated as described for assay method one. Some assays contained farnesol delivered in ethanol (or an ethanol only control) and were
preincubated for an additional 0-30 min prior to the addition of
labeled CDP-choline.
Metabolic Labeling--
CHO-K1 cells and CHO-K1 cell lines
containing inducible T7-CEPT1 were grown to mid log-phase in
Dulbecco's modified Eagle's media containing 5% fetal bovine serum
and 34 µg/ml proline. Doxycycline was added at 2 µg/ml for 24 h to induce T7-tagged CEPT1 expression. [14C]Choline (0.2 µCi) or [14C]ethanolamine (0.2 µCi) was added
simultaneously with the indicated concentration of farnesol. Subsequent
to incubation with radiolabel, cells were washed twice with ice-cold
phosphate-buffered saline and resuspended in 1 ml of methanol. Two ml
of chloroform and 1.5 ml of water were added, tubes were vortexed, and
then centrifuged at 2,500 × g for 10 min to facilitate
phase separation. Choline-containing aqueous phase metabolites were
separated in a solvent system consisting of CH3OH/0.6%
NaCl/NH4OH (50/50/5, v/v), and ethanolamine-containing metabolites were separated using CH3CH2OH/2%
NH4OH (1/2, v/v). Phospholipids in the organic phase were
routinely analyzed by thin layer chromatography on Whatman silica gel
60A plates using the solvent system
CHCl3/CH3OH/H20/CH3COOH
(70/30/4/2, v/v). Radiolabeled bands were detected using a BIOSCAN
System 200 imaging scanner, and appropriate bands were scraped into
scintillation vials for radioactivity determination.
Apoptosis Determinations--
Cells were washed with
Tris-buffered saline and disrupted by the addition of 1% Triton X-100
in Tris-buffered saline containing 1 mM EDTA and complete
protease inhibitor mixture (Roche Molecular Biochemicals) by incubation
at 4 °C for 10 min. The cell homogenate was centrifuged at
15,000 × g for 10 min at 4 °C to pellet unbroken cells and nuclei. Proteins were separated on a 10% SDS-polyacrylamide gel electrophoresis gel and transferred to polyvinylidene difluoride membranes using 25 mM Tris, 200 mM glycine, and
10% methanol for 2 h at 56 volts. Blots were blocked and probed
with a mouse monoclonal antibody toward PARP (1:2000), which recognizes
both the mature and caspase cleaved forms of the enzyme, and
Tris-buffered saline containing 0.1% Tween 20 (w/v) and 5% skim milk
powder. Blots were washed and then reprobed with a goat anti-mouse
secondary antibody coupled to horseradish peroxidase (1:10,000) in the
above buffer. Blots were washed and PARP protein was detected using the
ECL (Amersham Pharmacia Biotech) system.
The externalization of phosphatidylserine was monitored by annexin
V-fluorescein staining using the Annexin-V-FLUOS staining kit from
Roche Molecular Biochemicals and visualized by fluorescence microscopy.
Propidium iodide was used as a counterstain for nuclear DNA. The
manufacturer's method was used except that twice as much annexin V
conjugate was added to each sample as recommended.
Protein and Lipid Mass Determinations--
Protein was
determined by the method of Lowry et al. (24) using bovine
serum albumin as standard. Phospholipid phosphorus was determined by
the method of Ames and Dubin (25).
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RESULTS |
Effect of Farnesol on PC Synthesis--
Metabolic labeling
experiments have demonstrated that farnesol, a catabolite of the
cholesterol/isoprenoid pathway, dramatically inhibited PC synthesis at
the cholinephosphotransferase step in several cell types (8, 9, 19).
Previous work from our laboratory resulted in the isolation of the
first mammalian cholinephosphotransferase-encoding cDNA, human
choline/ethanolaminephosphotransferase 1 (CEPT1), whose corresponding
gene was expressed in similar levels in all tissues tested and that we
believe is a ubiquitous cholinephosphotransferase responsible for PC
synthesis in most tissues (21, 22). To further investigate the
interaction of farnesol with the CDP-choline pathway for PC synthesis
we established CHO cell lines capable of inducible over-expression of
CEPT1. Two separate CEPT1-inducible cell lines were constructed and
resulted in similar phenotypes and results for all studies presented,
although for the sake of brevity only one clone is represented in the
data shown. When the CEPT1 open reading frame was placed under the
control of a doxycycline-inducible promoter the addition of doxycycline
to the media resulted in a dramatic induction of CEPT1 protein as assessed by Western blot (Fig.
1A). An assay of
cholinephosphotransferase activity present in these CHO cells 24 h
after doxycycline treatment resulted in a 27-fold increase in
measurable cholinephosphotransferase activity (Fig. 1B).

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Fig. 1.
Development of stable cell lines capable of
inducible over-expression of T7-tagged hCEPT1. A,
Western blot probed with anti-T7 horseradish peroxidase-conjugated
monoclonal antibodies of 20 µg of CHO membrane protein isolated as
described under "Experimental Procedures" after incubation for
24 h with or without 2 µg/ml doxycycline. The arrow
indicates the CEPT1 band. B, in vitro
cholinephosphotransferase activity of the isolated protein samples
using the mixed micelle assay.
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Induced over-expression of CEPT1 did not increase the rate of PC
synthesis (Fig. 2, B and
C). This is not surprising as the cholinephosphotransferase
step is normally not rate-limiting for PC synthesis (10-14). The
treatment of uninduced CHO-K1 cells with farnesol resulted in a
dramatic inhibition of PC synthesis to levels ~30% of control at
4 h, the limit of our time course study (Fig. 2B). An
analysis of radiolabel in the metabolites of the Kennedy pathway for PC
synthesis indicated two main alterations; the first was a dramatic
decrease in label associated with the phosphocholine fraction
indicative of an up-regulation of CTP:phosphocholine cytidylyltransferase, and the second was a massive increase in radiolabel associated with the CDP-choline fraction. As PC synthesis was ultimately inhibited the metabolic block at the
cholinephosphotransferase step was the over-riding factor that resulted
in an inhibition of PC synthesis. This observation is consistent with
other studies with respect to the inhibition of PC synthesis by
farnesol (8, 9, 19). Upon induced over-expression of the CEPT1 protein we were able to completely reverse farnesol-induced inhibition of PC
synthesis (Fig. 2C). An analysis of the radiolabeled
Kennedy pathway metabolites revealed several interesting
regulatory processes. The radiolabel associated with the phosphocholine
fraction remained low even though the rate of PC synthesis had returned
to normal levels indicating that the CTP:phosphocholine
cytidylyltransferase step was still up-regulated, implying farnesol may
irreversibly activate CTP:phosphocholine cytidylyltransferase. The
CDP-choline level remained high implying that although the increased
cholinephosphotransferase activity provided by induction of CEPT1 was
able to successfully convert CDP-choline into PC, activation of the
upstream CTP:phosphocholine cytidylyltransferase step kept the label
associated with CDP-choline fraction high.

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Fig. 2.
Effect of farnesol on PC biosynthesis.
A, the Kennedy pathway for the synthesis of PC.
B, radiolabeled choline was added to uninduced CHO-K1-CEPT1
cells simultaneously with 80 µM farnesol (closed
symbols) or an ethanol control (open symbols). PC and
the Kennedy pathway metabolites were separated and associated label was
quantitated as described under "Experimental Procedures."
C, CHO-K1-CEPT1 cells were induced to over-express CEPT1 for
24 h with 2 µg/ml doxycycline and were subsequently treated with
80 µM farnesol (closed symbols) or an ethanol
control (open symbols). PC and the Kennedy pathway
metabolites were separated and associated label was quantitated as
described under "Experimental Procedures."
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This study illustrates that although farnesol has pleiotropic effects
within the Kennedy pathway for PC synthesis, farnesol-induced inhibition of PC synthesis is manifested at the level of the
cholinephosphotransferase step. This study also provides the first data
that describes the ability of the dual specificity CEPT1
choline/ethanolaminephosphotransferase to supply
cholinephosphotransferase activity for the synthesis of PC in mammalian
cells (20, 21).
Effect of CEPT1 on Farnesol-induced Apoptosis--
Genetic
inactivation of PC synthesis via the use of a
temperature-sensitive allele of CTP:phosphocholine
cytidylyltransferase, the step that is normally rate-limiting for PC
synthesis, resulted in apoptosis in CHO cells (15). The addition of
farnesol to several cell types also induced apoptosis and correlated
with the ability of farnesol to inhibit PC synthesis at the
cholinephosphotransferase step (8, 9, 19). Farnesol-induced apoptosis
was partially rescued by the addition of diacylglycerol or PC, but not
other lipids (8, 9, 20). In our study in CHO cells farnesol induced
apoptosis, as monitored by cleavage of PARP and the externalization of
phosphatidylserine at 80 µM and above (Fig.
3, A and B). This was a similar concentration to that observed for other cell types (8,
9, 19). A close examination of the phosphatidylserine externalization
data (Fig. 3B) indicates that an estimated 80% of the cells
have externalized their phosphatidylserine, whereas ~15% also have
their nucleus stained with propidium iodide implying that these cells
are either in a much later stage of apoptosis or have undergone
necrosis. We are in the process of isolating this cell population using
fluorescence cell sorting to determine their mode of death. The most
notable observation from this set of experiments was that CHO cells
induced to over-express CEPT1, a condition that we demonstrated
restored PC synthesis to normal levels (Fig. 2), did not result in a
rescue of farnesol-induced cell death (Fig. 3, A and
B). This implies that direct inhibition of
cholinephosphotransferase by farnesol may not be the mechanism by which
farnesol induced apoptosis (9).

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Fig. 3.
Effect of over-expression of CEPT1 on
farnesol-induced apoptosis. A, uninduced CHO-K1-CEPT1
cells were incubated in increasing concentrations of farnesol for
3 h. Cell extracts were probed with anti-PARP antibodies that
detect the uncleaved (116 kDa) and caspase cleaved (85 kDa) forms of
PARP. B, CHO-K1-CEPT1 cells induced to over-express CEPT1
for 24 h with 2 µg/ml doxycycline prior to a 3-h farnesol
incubation. Cell extracts were probed with anti-PARP antibodies that
detect the uncleaved (116 kDa) and caspase cleaved (85 kDa) forms of
PARP. "I" indicates induced cells and "C"
indicates uninduced control cells. C, CHO-K1-CEPT1 cells
induced to over-express CEPT1 for 24 h with 2 µg/ml doxycycline
prior to a 3-h farnesol incubation. Cells were stained with annexin
V-fluorescein or propidium iodide and visualized as described under
"Experimental Procedures." Merged figures are on overlap of the
annexin V-fluorescein and propidium iodide panels.
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Effect of Farnesol on in Vitro Cholinephosphotransferase
Activity--
The ability of farnesol to induce apoptosis through the
inhibition of PC synthesis was hypothesized to work via
direct competitive inhibition of cholinephosphotransferase enzyme
activity for diacylglycerol by farnesol (9). This was demonstrated
using subcellular membranes as a source of cholinephosphotransferase
enzyme and an enzyme assay that delivered the insoluble diacylglycerol
and farnesol as an emulsion using either a very small amount of
detergent or without the aid of detergent solubilizers (8, 9). We also observed that the greatest in vitro inhibition of
cholinephosphotransferase activity involved preincubation of the
cellular membranes with farnesol (8). We also observed a significant
inhibition of cholinephosphotransferase activity by farnesol, to 32%
control values, when the lipid substrates were delivered without the
aid of solubilizing detergents (Table I).
Preincubation of the assay mixture with farnesol for 30 min prior to
the addition of the radiolabeled CDP-choline substrate resulted in a
cholinephosphotransferase activity that was 6% of control levels.
Because our metabolic labeling and induction of apoptosis data
seemingly uncoupled farnesol-induced apoptosis from the inhibition of
PC synthesis we hypothesized that under the conditions used in the
previous analysis of cholinephosphotransferase activity farnesol may be
progressively altering the membrane structure such that the integral
membrane cholinephosphotransferase protein was becoming inactivated
(26). To test this possibility and to further characterize the ability
of farnesol to inhibit cholinephosphotransferase activity we developed
a mixed micelle cholinephosphotransferase enzyme assay. The mixed
micelle method results in a uniform delivery of lipid-soluble
substrates and activators/inhibitors and eliminates the ability of the
lipids to alter the bulk membrane environment (27). When
cholinephosphotransferase activity was monitored using a mixed micelle
assay there was no inhibition by farnesol or phosphorylated derivatives
of farnesol or geranylgeranyl present downstream of farnesol in the
isoprenoid pathway (Table II).
Preincubation of the assay mixture with farnesol also did not result in
an inhibition of cholinephosphotransferase activity. To ensure the
mixed micelle assay condition used is an environment compatible with
accurately measuring cholinephosphotransferase activity we tested if a
previously identified inhibitor of cholinephosphotransferase,
chelerythrine, was able to inhibit cholinephosphotransferase activity.
Chelerythrine at 50 µM inhibited
cholinephosphotransferase activity to 38% control values and at 100 µM to 25% control values when assayed using our mixed
micelle system; this was a similar level of inhibition to that observed
using an emulsion-based assay (8).
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Table II
Effect of farnesol and its analogues and metabolites on
cholinephosphotransferase activity using a mixed micelle enzyme assay
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In the studies that had delivered lipids as emulsions or without the
aid of detergents and had demonstrated cholinephosphotransferase inhibition by farnesol the net concentration of diacylglycerol was 2 mM, and the amount of farnesol required to observe
significant cholinephosphotransferase activity inhibition was 60-80
µM. In our mixed micelle system diacylglycerol was
delivered as 10 mol% lipid, which corresponds to an effective
concentration of 1.2 mM, and farnesol was added at 100 µM or 0.8 mol%. Hence, our effective ratio of farnesol
to diacylglycerol was greater than that used previously to
demonstrate enzyme activity inhibition. We predict that in
vitro, farnesol likely does not directly inhibit
cholinephosphotransferase activity but instead can compromise the
structure of the membrane used as enzyme source and progressively
inactivate the enzyme.
Effect of Diacylglycerol on Farnesol-induced
Apoptosis--
Diacylglycerol is one of the substrates for
cholinephosphotransferase, and administration of diacylglycerol to
cells in culture was found to partially prevent farnesol-induced
apoptosis (9, 20). This was one of the observations that led to the
hypothesis that farnesol induced apoptosis by direct inhibition of
cholinephosphotransferase activity and subsequent limitation of PC
synthesis. Our results demonstrate that annexin V externalization was
dramatically decreased upon addition of di18:1 diacylglycerol to
farnesol-treated cells indicating protection versus
apoptosis by diacylglycerol (Fig. 4). A
close inspection of the results indicated that there was only a small
decrease in cells whose nucleus was stained with propidium iodide
suggesting that the subset of cells stained positive by both annexin V
and propidium iodide may be undergoing apoptosis as opposed to
necrosis, and diacylglycerol can only rescue the apoptotic cells.
Definitive evidence will require isolation of this specific population
of cells and an analysis of their mode of death.

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Fig. 4.
Effect of exogenous diacylglycerol on
farnesol-induced apoptosis. CHO-K1 cells were incubated with 80 µM farnesol or an equal volume of ethanol delivery
vehicle for 3 h ± 40 µg/ml di18:1 diacylglycerol. Cells were
stained with annexin V-fluorescein and propidium iodide and visualized
as described under "Experimental Procedures." Merged figures are on
overlap of the annexin V-fluorescein and propidium iodide panels.
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We also observed the effect of exogenous administration of
diacylglycerol on farnesol-induced inhibition of PC synthesis. The
addition of diacylglycerol was unable to restore PC synthesis to
control levels as monitored by the incorporation of radiolabeled choline into PC and corresponding upstream metabolites of the Kennedy
pathway (Fig. 5). Thus, diacylglycerol
rescue of farnesol-mediated apoptosis is not via restoration
of de novo PC synthesis.

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Fig. 5.
The effect of farnesol and exogenous
diacylglycerol on PC metabolism. CHO-K1 cells were incubated with
80 µM farnesol (F) or an equal volume of
control ethanol delivery vehicle for 4 h ± 40 µg/ml di18:1
diacylglycerol. The samples were then analyzed for choline metabolites
as described under "Experimental Procedures." Results are expressed
as the means ± S.E. of three separate experiments.
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Farnesol Inhibition of Phosphatidylethanolamine Synthesis--
To
test if farnesol inhibition of PC synthesis was specific to the Kennedy
pathway for the synthesis of PC, we tested whether farnesol could also
inhibit the analogous pathway for the synthesis of PE (28). Treatment
of CHO cells with farnesol inhibited the synthesis of PE as monitored
metabolically by the addition of radiolabeled ethanolamine to the
medium of cells concomitant with farnesol treatment (Fig.
6). Analysis of the metabolites within the CDP-ethanolamine pathway indicated increases in label associated with ethanolamine and phosphoethanolamine, consistent with inhibition at the CTP:phosphoethanolamine cytidylyltransferase step of this pathway. This was in contrast to that observed for the Kennedy pathway
for the synthesis of PC in that phosphocholine labeling decreased and
CDP-choline labeling increased upon treatment with farnesol. Induction
of CEPT1, which can synthesize both PC from CDP-choline and PE from
CDP-ethanolamine in vitro and in vivo and is the
only ethanolaminephosphotransferase present in mammalian cells (21,
22), was unable to rescue farnesol-induced inhibition of PE synthesis
(Fig. 6B). Thus, farnesol-induced inhibition of phospholipid
synthesis is pleiotropic and not limited to either the inhibition of PC
synthesis or the final phosphotransferase reaction of the Kennedy
pathways.

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Fig. 6.
Effect of farnesol and CEPT1 over-expression
on PE biosynthesis. A, radiolabeled ethanolamine was
added to CHO-K1 cells simultaneously with 80 µM farnesol
(closed symbols) or an ethanol control (open
symbols). PE and the biosynthetic pathway metabolites were
separated and associated label was quantitated as described under
"Experimental Procedures." B, CHO cells induced to
over-express CEPT1 for 24 h with 2 µg/ml doxycycline were
treated with 80 µM farnesol (closed symbols)
or an ethanol control (open symbols). PE and the pathway
metabolites were separated, and associated label was quantitated as
described under "Experimental Procedures."
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DISCUSSION |
Farnesol is a catabolite of the cholesterol/isoprenoid
biosynthetic pathway whose administration preferentially induces
apoptosis in transformed versus untransformed cells
(16-18). The intracellular phosphorylation of farnesol results in the
synthesis of farnesol pyrophosphate, which allows for the incorporation
of farnesol directly into proteins most notably small G proteins
including Ras (29, 30). This protein prenylation event is an essential process for oncogenic Ras to affect cellular transformation, and the
farnesyl:protein transferase step is the target of several new
anticancer drugs (31, 32). The ability of farnesol administration to
alter the prenylation of the small G proteins Ras and Rap1A was
tested and neither the prenylation event nor the ability of the G
proteins to associate with the membrane was altered (9). Thus, it
appears that farnesol administration preferentially kills transformed
cells by a mechanism independent of protein prenylation events.
The addition of farnesol and several other compounds that induce
apoptosis resulted in the preferential accumulation of the metabolite
CDP-choline as monitored by NMR spectroscopy (7). CDP-choline is used
in conjunction with diacylglycerol to synthesize PC by the enzyme
cholinephosphotransferase. As genetic inactivation of PC synthesis in
CHO cells resulted in apoptosis (15), it was postulated that farnesol
induced apoptosis through its repression of PC synthesis by direct
inhibition of cholinephosphotransferase. Evidence gathered in support
of this hypothesis has been substantial and includes the observations
that (i) metabolic labeling of cells in culture resulted in a dramatic
decrease in PC labeling with a corresponding increase in CDP-choline
labeling (8, 9, 19), (ii) farnesol-induced apoptosis could be prevented
by the addition of diacylglycerol or PC but not other lipids to the
media of cells (8, 9, 20), and (iii) farnesol inhibited
cholinephosphotransferase enzyme activity in vitro (8,
9).
To further examine the precise interaction between farnesol-induced
apoptosis and the inhibition of PC synthesis at the
cholinephosphotransferase step we constructed CHO cell lines capable of
inducible over-expression of CEPT1, a dual specificity human
choline/ethanolaminephosphotransferase capable of synthesizing PC and
PE in vitro and in vivo (21, 22). Over-expression
of CEPT1 prevented farnesol-induced inhibition of PC synthesis but did
not prevent farnesol-induced apoptosis. This was the first
demonstration that CEPT1 could synthesize PC in mammalian cells and
also implied that inhibition of de novo PC synthesis may not
be the mechanism by which farnesol causes apoptosis. We demonstrated
that the addition of diacylglycerol, one of the substrates of the
cholinephosphotransferase enzyme, prevented farnesol-induced apoptosis
(8, 9, 20), but most notably exogenous diacylglycerol administration
did not reconstitute PC synthesis. The most likely explanation for the
inability of diacylglycerol to restore PC synthesis, yet prevent
farnesol-induced apoptosis, is that farnesol causes apoptosis by
inhibiting a diacylglycerol-mediated process that is produced at an
intracellular site distinct from the diacylglycerol pool used for PC synthesis.
To further study the mechanism by which farnesol affected PC synthesis
we devised a mixed micelle method for the determination of CEPT1
cholinephosphotransferase enzyme activity (27). This was in contrast to
the assays used previously to demonstrate the inhibition of
cholinephosphotransferase activity by farnesol, which delivered lipids
as an emulsion or did not include lipid-solubilizing detergents in the
assay mixture (8, 9). The mixed micelle method solubilizes the membrane
used as enzyme source such that the addition of exogenous lipid
substrates or activators/inhibitors does not alter the physical
parameters of the micelle containing the cholinephosphotransferase
enzyme (27). This is not the case for the assay in which farnesol was
delivered as in emulsion using a small amount of detergent or without
the aid of detergents (8, 9). Using the mixed micelle assay neither
farnesol nor its phosphorylated derivatives inhibited CEPT1
cholinephosphotransferase activity. We conclude from this study that
farnesol likely does not directly inhibit cholinephosphotransferase
activity in vitro but instead alters the physical properties
of the membrane such that when farnesol is delivered in the absence of
excess detergent cholinephosphotransferase is progressively
inactivated. However, the issue of precisely how farnesol inhibits
cholinephosphotransferase remains to be resolved. Acidification of the
cytosol is associated with most forms of apoptosis and Anthony et
al. (8) demonstrated that farnesol rapidly acidified the cytosol
in a time dependent manner consistent with inactivation of
cholinephosphotransferase activity, especially because the active site
of cholinephosphotransferase is known to face the cytoplasm. However,
our over-expression of CEPT1 rescued PC synthesis when cells were
challenged with farnesol, and acidification should inactivate an enzyme
regardless of its expression level. The same explanation could be used
to discount the possibility that farnesol physically perturbs the
biological membrane resulting in an inhibition of
cholinephosphotransferase activity. However, there are few other known
effectors of cholinephosphotransferase activity that could be overcome
by an increase in CEPT1 expression. One of these is a decrease in
cellular diacylglycerol mass, which has been demonstrated to result in
the cholinephosphotransferase step becoming rate-limiting for PC
synthesis (33, 34), and the other is a strong in vitro
inhibition of cholinephosphotransferase by Ca2+ (35).
Neither of these parameters has been measured subsequent to farnesol
addition, but both are dependent on a molar amount to alter
cholinephosphotransferase activity and could be overcome by increased
expression of CEPT1.
The ability of diacylglycerol and PC but not other lipids to prevent
farnesol-induced apoptosis (8, 9, 20), together with the data presented
in our study, leads one to speculate that farnesol or one of its
metabolites prevents a diacylglycerol-mediated event that is derived
from PC and required for cell survival. If true, a farnesol-mediated
process could either interfere with the generation of diacylglycerol
produced from PC and/or affect a diacylglycerol target molecule that
produces a cell survival signal. Indeed, the diacylglycerol regulated
protein kinase C
and protein kinase C
isoforms have been
demonstrated to translocate from their active membrane-bound forms to
their storage cytosolic forms upon farnesol administration (36,
37).
The precise pathway by which farnesol causes apoptosis needs to be
determined in further detail to delineate how farnesol preferentially
targets transformed cells versus those that are untransformed for cell death (17). Our study has clarified some of the
mechanisms of cross-talk between various lipid metabolic pathways and
has highlighted their importance in the balance between the generation
of cell survival and apoptotic messages.