(Received for publication, October 31, 1994; and in revised form, December 27, 1994)
From the
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), 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
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
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.
PtdCho ()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. LPC and ET-18-OCH
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 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
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
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
-treated cells
have reduced levels of PtdCho. Subsequently, several groups showed that
ET-18-OCH
decreases choline incorporation into PtdCho (37, 38, 39, 40) . The mechanism for
inhibition of this pathway has not been established although
ET-18-OCH
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.
For the experiments reported in
this paper, either LPC or ET-18-OCH was added followed by
[
H]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 10
cells/60-mm dish with 50
µM egg LPC plus [1-
C]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.
Figure 2:
LPC inhibited
[H]choline incorporation into phospholipid.
BAC1.2F5 cells were grown for 2 days to a density of 2.8
10
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
(Panel B) were added. Five min later,
[
H]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 [
H]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
[H]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 [
H]choline-derived metabolites
reflected the actual distribution of the PtdCho precursors. LPC had a
small inhibitory effect on the total [
H]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
[H]choline uptake by BAC1.2F5 cells in the
presence and absence of LPC. Cells were grown to a density of 6.5
10
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, [
H]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 [
H]choline
incorporated into the cells (Panel A). The cell pellets were
extracted to determine the amounts of [
H]choline
incorporated into the phospholipid (Panel B) or soluble (Panel C) fractions as described under ``Experimental
Procedures.'' Untreated cells (
); LPC-treated cells (
).
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 [H]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 (); LPC-treated
cells (
).
An identical series of experiments and
data analysis was carried out with BAC1.2F5 cells treated with 12
µM ET-18-OCH. In both cases, PtdCho synthesis
was effectively blocked. Choline and phosphocholine accumulated in
ET-18-OCH
-treated cells up to 2 h, but then decreased at
the 4- and 6-h time points. ET-18-OCH
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
-treated and LPC-treated
cells was attributed to the cytotoxicity of ET-18-OCH
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
(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
[H]choline into BAC1.2F5 cells (Fig. 5).
LPS and LPE had no significant effect on the amount of
[
H]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
10
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,
[
H]choline (1.5 µCi/ml, 2 µM final concentration) was added and the cells were incubated for 4
h. The extent of [
H]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 by the transient
removal of CSF-1 (49) and then growth factor was added along
with [
H]thymidine and increasing concentrations
of LPC (0-200 µM). After 24 h of incubation, the
maximum decrease in [
H]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
blocked [
H]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.
Figure 6:
Neither LPC nor ET-18-OCH altered the cellular content of CT. BAC1.2F5 cells were grown to
a density of 9
10
cells/100-mm dish and treated
with either 100 µM LPC or 12 µM ET-18-OCH
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 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
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
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
-treated cells.
Figure 7:
Effect of LPC and ET-18-OCH 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
(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
-treated, 228.6 ±
12.
Figure 8:
LPC and
ET-18-OCH 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
. Panel D, kinetic analysis of CT
inhibition by ET-18-OCH
. 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
concentrations
was analyzed using double-reciprocal plots. Data points are the average
of duplicate reactions from a representative
experiment.
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 ( Fig. 2and Fig. 8). ET-18-OCH
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
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
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 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
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
-treated
cells (Fig. 7). ET-18-OCH
is known to partition into
biological membranes (59) and the association of CT with the
particulate fraction of ET-18-OCH
-treated cells is
consistent that the idea that ET-18-OCH
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
-treated cells. Our data are not consistent with
the report of Tronchere et al.(40) who concluded that
ET-18-OCH
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
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
. Thus, it remains
possible that CT is localized in the particulate fraction of
ET-18-OCH
-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
-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. 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
inhibition of CT as competitive with respect to
lipid activators, suggesting that the biological effects of
ET-18-OCH
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
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.