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J. Biol. Chem., Vol. 275, Issue 28, 21197-21202, July 14, 2000
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From the Department of Biochemistry and Canadian Institutes of
Health Research Group on Molecular and Cell Biology of Lipids,
University of Alberta, Edmonton, Alberta T6G 2S2, Canada
Received for publication, April 26, 2000, and in revised form, May 3, 2000
The mutant Chinese hamster ovary cell
line (CHO), MT58, has a temperature-sensitive mutation in
CTP:phosphocholine cytidylyltransferase (CT), preventing
phosphatidylcholine (PC) synthesis at 40 °C which results in
apoptosis. Previous studies (Houweling, M., Cui, Z., and Vance, D. E. (1995) J. Biol. Chem. 270, 16277-16282) showed that expression of wild-type CT- Phosphatidylcholine
(PC)1 is the most abundant
phospholipid in mammalian cellular membranes. Besides having a
structural role in membranes and lipoproteins, PC plays an important
role in signal transduction as it is a major source of lipid second
messengers (1). In all nucleated cells, PC is made primarily through
the CDP-choline pathway in which the key enzyme is CTP:phosphocholine cytidylyltransferase (CT) (2). However, in hepatic cells, an alternative pathway exists utilizing phosphatidylethanolamine N-methyltransferase (PEMT), which converts
phosphatidylethanolamine (PE) to PC via three sequential methylation
events (3).
There are several reasons why hepatic cells may maintain two pathways
for providing PC. One possibility is that PEMT is necessary for the
endogenous production of choline, which may have several fates:
including biosynthesis of acetylcholine, a source for betaine (4) and
conversion to PC by the CDP:choline pathway. Other studies have
suggested that PEMT-derived PC may be preferentially secreted with
lipoproteins (5). Recent studies with the PEMT knockout mouse indicate
that the PEMT gene may have been conserved during evolution, to provide
PC when dietary choline levels are insufficient during such times as
pregnancy or starvation (6). Curiously, previous studies indicated that
PC generated via the PEMT pathway did not substitute for PC derived
from the CDP:choline pathway in cells that are defective in CT (7).
Esko and co-workers (8) first described the mutant Chinese hamster
ovary (CHO) cell line, MT58, which has a temperature-sensitive mutation
in CT. These cells divide normally at 33 °C, but at 40 °C CT
activity is diminished, PC levels are decreased and the cells die via
apoptosis (7, 9). Using this cell line, Houweling and co-workers (7)
showed that at the restrictive temperature (40 °C), cDNA
encoding CT- There are several hypotheses as to why PEMT-derived PC does not rescue
the mutant cell line, including subcellular location and differences in
the molecular species of PC produced by the two pathways. We
investigated these and other hypotheses and have determined the rate of
PC degradation is greater than the rate of PC synthesis in the
PEMT2-expressing MT58 cells grown at 40 °C. Therefore,
PEMT2-expressing MT58 cells do not produce enough PC to maintain
cellular replication after 24 h at 40 °C
Materials--
Ham's F-12 medium, fetal bovine serum (FBS), and
G418 were from Life Technologies, Inc., and culture dishes and flasks
were from Beckton Dickinson. Silica gel G60 plates were obtained from Merck. [methyl-3H]Choline chloride,
[methyl-3H]methionine, and
[1-3H]ethanolamine were obtained from Amersham Pharmacia
Biotech. Methyl arachidonyl fluorophosphonate, manoalide, and D609 were obtained from Calbiochem. L-659989, a platelet-activating
factor receptor antagonist, which inhibits phospholipase D, was a
generous gift from Merck Frost. Lyso-PC (LPC), alkyl-LPC, and DOTAP
were purchased from Avanti Polar Lipids. All other chemicals and
reagents were obtained from standard commercial sources.
Cell Culture and Growth Curves--
Wild-type and MT58 cell
lines were cultured in Ham's F-12 medium supplemented with 10% FBS.
PEMT2-expressing and CT-expressing MT58 cell lines (7) were cultured in
Ham's F-12 medium supplemented with 10% FBS and 0.3 mg/ml G418. All
cells were maintained in 75-mm culture flasks at 5% CO2,
90% relative humidity at 33 °C. For growth curves, cells were
plated at 2.5 × 105 in 60-mm dishes containing 4 ml
of medium and incubated at the indicated temperature. At the indicated
times, culture medium was removed, the adherent cells were harvested
with trypsin and viable cells that excluded trypan blue were counted.
Where indicated, cells were also grown in the presence of delipidated
FBS or phospholipase inhibitors.
Transfection of PEMT-expressing MT58 cells--
PEMT-expressing
MT58 cells were plated at 2 × 105 cells/60-mm dish
and grown overnight. Transfection with the PEMT2 expression vector or
the empty vector (7) was performed the next day via a DOTAP
transfection method (10). DOTAP was dried under vacuum and resuspended
in sterile water to a concentration of 1 mg/ml. 15 µl of the DOTAP
solution and 0.5 µg of DNA were incubated at room temperature with
serum-free medium (final volume, 250 µl) for 20 min. During this
time, the dishes were washed with serum-free medium and 3 ml of
serum-free medium was added to each dish. The liposome-DNA suspension
was then added dropwise to the dishes (10, 11). After 4 h, the
medium was removed and replaced with growth medium and the cells were
grown at 40 °C. At the indicated times, the culture medium was
removed, the adherent cells were harvested with trypsin, and viable
cells that excluded trypan blue were counted.
Incorporation of Radiolabeled Precursors into PC--
For
in vivo PEMT activity analysis, PEMT2-expressing MT58 cells
were grown to 40% confluence and then shifted to 40 °C. At the
indicated times, cells were labeled with [3H]methionine
(5 µCi/dish) for 2 h and then harvested in phosphate-buffered saline (PBS) as described below. For ethanolamine, oleate, and choline
labeling experiments, cells were first incubated with the appropriate
radiolabel ([3H]oleate, [3H]ethanolamine or
[3H] choline, respectively) at 5 µCi/dish at 33 °C
for 24 h. The medium was removed from the cells, fresh medium was
added, and the cells were incubated at 40 °C. At the indicated
times, cells were washed three times with cold PBS and harvested in 10 ml of PBS. The cells were pelleted by low speed centrifugation,
resuspended in PBS, and sonicated. Lipids were extracted as described
by Sundler and co-workers (12) and separated by thin layer
chromatography (TLC) on Silica gel G60 plates as described below. For
analysis of lyso-PC (LPC), phospholipids were separated with
chloroform/methanol/acetic acid/formic acid/water (70:30:12:4:1) as a
developing solvent. After TLC, LPC was visualized by iodine vapor and
the radiolabel incorporated into the bands of interest was measured by
liquid scintillation counting after being scraped off the plate.
Determination of Phospholipid Mass, Triacylglycerol (TG) Mass,
and Protein--
Cells were cultured and harvested as described above
except radiolabeled precursors were omitted. Lipids from 0.5 mg of
protein were extracted as described previously. For analysis of PC,
phospholipids were separated using TLC with chloroform/methanol/acetic
acid/formic acid/water (70:30:12:4:1) as a developing solvent. For TG
analysis, TLC using heptane/diisopropyl ether/acetic acid (60:40:4) as
the developing solvent separated neutral lipids. After TLC, and
visualization with iodine vapor, the bands of interest were scraped and
analyzed. PC mass was determined by measuring the phosphorous content
(13), and TG mass was determined by the hydroxylamine method described by Snyder and Stephens (14). Protein concentrations were determined using the Coomassie Plus protein protocol from Pierce, which is based
on the Bradford method (15). Bovine serum albumin was used as a standard.
Changing the Medium Rescues PEMT2-expressing MT58 Cells at
40 °C--
We investigated, without success, a variety of
hypotheses as to why PEMT-derived PC does not rescue the mutant cell
line. First, we analyzed the contribution of the PEMT pathway and
CDP-choline pathway in generating nuclear PC. Using radiolabeling
studies, we found that the percent of PC levels derived from the PEMT
pathway or the CDP-choline pathway was similar in PEMT2-expressing MT58 and CT-expressing MT58 cells (3.1% and 3.3% of total choline
containing phospholipids, respectively). Next, we determined if there
were any alterations in downstream signaling by PC derived second
messengers in PEMT-expressing MT58 cells compared with wild-type and
CT-expressing MT58 cells. We observed no differences in levels of
protein phosphorylation, particularly tyrosine phosphorylation and
mitogen-activated protein kinase activation.
Differences in the molecular composition of the PC species from the two
pathways were also examined. Using gas chromatography, we found
negligible differences in the fatty acid composition of PC derived from
the PEMT-expressing MT58 cells compared with PC derived from
CT-expressing MT58 cells grown at 40 °C (data not shown).
Furthermore, we found that alkyl-LPC could rescue the PEMT-expressing
MT58 cells grown at 40 °C (22 ± 5 × 105
cells/dish for PEMT-expressing MT58 cells; 32 ± 6 × 105 for wild-type cells after 96 h, n = 6). These data suggest that potential differences in the molecular
composition of PC created by the PEMT pathway are not the reason why
PEMT2 does not rescue MT58 cells.
In the process of performing the above experiments, we observed that
the PEMT2-expressing MT58 cells could be rescued at 40 °C simply by
changing the growth medium every 24 h. Fig.
1 shows that at 40 °C the
PEMT2-expressing MT58 cells divide once and then fail to replicate as
described earlier (7). When the medium was replaced every 24 h
with medium containing serum, the PEMT2-expressing MT58 cells had a
growth rate comparable to those cells grown at 33 °C as well as
wild-type cells grown at 40 °C (32 ± 6 × 105
wild-type cells/dish after 96 h, n = 6). This was
not due to the cellular uptake and utilization of serine, ethanolamine,
or methionine, precursors to PE that could then be methylated to PC,
since supplementing the medium with these compounds every 24 h did
not rescue the cells (data not shown).
Delipidated Serum Does Not Support the Growth of PEMT2-expressing
MT58 Cells at 40 °C--
Subsequently, we incubated PEMT-expressing
MT58 cells with medium containing delipidated serum at 40 °C (Fig.
2). Fig. 2A shows that
PEMT2-expressing MT58 cells failed to undergo replication when grown at
40 °C with medium containing delipidated serum, even when the medium
was changed every 24 h. This was not due to the loss of a growth
factor during the delipidation process, as medium containing
delipidated serum did not retard the growth of the PEMT2-expressing
MT58 cells at 33 °C (Fig. 2B). When the delipidated serum
was supplemented with either LPC or lyso-platelet-activating factor
(alkyl-LPC), we found that PEMT2-expressing MT58 cells divided at the
restrictive temperature yielding 20 × 105 and 22 × 105 cells/dish at 96 h, respectively
(n = 2). Taken together, these data suggested that PC
levels might not be sufficient in the PEMT2-expressing MT58 cells for
continued growth at 40 °C.
PC Levels Are Not Maintained in PEMT2-expressing MT58 Cells Grown
at 40 °C--
Based on the above data, we next measured PC levels
in four CHO cell lines: wild-type, MT58, CT-expressing MT58, and
PEMT2-expressing MT58 cells (7). At 33 °C in all four cell lines, PC
content varied little over 72 h (~70 nmol of PC/mg of protein).
When the cells were shifted to 40 °C, we found that the wild-type
and CT-expressing MT58 cells increased their PC content by 2- and
1.7-fold, respectively, over 72 h (Fig.
3A). In contrast, MT58 cells
did not have an increase in PC content once shifted to the restrictive
temperature, as previously reported (7, 8). Initially, PC levels in the PEMT2-expressing cell lines were maintained at 24 h. However, the
PEMT2-expressing MT58 cells also did not have an increase in PC over
time (Fig. 3A, triangles) when compared with the
wild-type and CT-expressing cell lines.
PEMT Activity Is Maintained in PEMT2-expressing MT58 Cells at
40 °C--
Possible explanations for the lack of increase in PC
levels once the cells were shifted to the restrictive temperature were either that the PEMT2 enzyme was no longer functional or that less
PEMT2 was present at the restrictive temperature. We found that there
was no change in the amount of immunoreactive PEMT2 after 72 h at
40 °C (data not shown). Therefore, decreased amounts of the protein
did not account for the lack of PC. We next determined if the PEMT2
protein was active at 40 °C over the 72-h time course. Fig.
3B shows that the incorporation of radiolabeled methionine into PC occurs at 40 °C, indicating PEMT activity. In fact, at 40 °C, methionine incorporation is severalfold higher than at 33 °C. Therefore, the PEMT2 protein is still functional after 72 h at 40 °C.
PEMT2-expressing MT58 and MT58 Cells Accumulate TG at
40 °C--
When analyzing the PC content in the above experiments,
we noticed that there was a prominent band during TLC in the neutral lipids from PEMT2-expressing MT58 cells and MT58 cells at 40 °C. Upon further investigation, we determined that this was due to TG
accumulation. Fig. 4 shows that, at
40 °C, TG accumulated in wild-type and CT-expressing MT58 cells,
3.7- and 3.8-fold, respectively. This accumulation, however, was
similar to the levels of all four cell lines grown at 33 °C (Fig.
4). In contrast, at 40 °C there was a much greater increase in TG
accumulation in both the mutant and PEMT2-expressing MT58 cells, 10- and 16-fold increase, respectively (Fig. 4). At 40 °C, the
PEMT2-expressing MT58 cells accumulated approximately 1.6-fold more TG
than MT58 cells after 72 h (compare closed
triangles to closed diamonds).
Recently, studies have shown that phospholipase A2 plays a
role in regulating cellular PC levels (16, 17). Based on these studies,
we analyzed the LPC levels in both PEMT2-expressing MT58 cells and
CT-expressing MT58 cells. PEMT2-expressing MT58 cells were labeled with
[3H]ethanolamine, which would be expected to result in
labeled PC by methylation of PE by PEMT. CT-expressing MT58 cells were
labeled with [3H]choline. After 24 h at 40 °C,
labeled LPC levels were 2-fold higher in PEMT2-expressing MT58 cells
compared with the CT-expressing MT58 cells, and they remained
1.5-2-fold higher over 72 h at 40 °C. On the basis of this
result, we hypothesized that at 40 °C the rate of PC synthesis was
less than the rate of PC degradation in the PEMT2-expressing and MT58
cells when compared with the wild-type and CT-expressing MT58 cells. If
this were indeed the case, we hypothesized that PEMT2-expressing MT58
cells may be rescued by stimulating PC synthesis or by inhibiting PC degradation.
Transfection of PEMT2 cDNA Partially Rescues PEMT2-expressing
MT58 Cells at 40 °C--
We attempted to increase PC synthesis in
the PEMT-expressing MT58 cells, at 40 °C, by transfecting these
cells with additional PEMT2 cDNA. Fig.
5 shows that transient transfection of
0.5 µg of PEMT2 containing vector increased PEMT2-expressing MT58
cell growth at 40 °C compared with empty vector-transfected
controls. Transfection with additional PEMT2 cDNA increased the
number of cells per dish at 72 h after transfection compared with
control cells, 11 × 105 and 3.4 × 105 cell/dish, respectively (Fig. 5). This result suggested
that, by shifting the synthesis/degradation equilibrium in favor of PC
synthesis, PEMT2-expressing MT58 cells were able to replicate at
40 °C.
Phospholipase Inhibitors Partially Rescue the Growth of
PEMT2-expressing MT58 Cells at 40 °C--
We next determined if
inhibition of PC degradation would rescue the PEMT2-expressing MT58
cells using four phospholipase inhibitors. D609 is a PC phospholipase C
inhibitor that has also been shown to inhibit phospholipase D (18, 19).
Manoalide and MAFP are phospholipase A2 inhibitors
(20-22), and L-659989 (Merck Frost) is a platelet-activating factor
receptor antagonist that has been shown to inhibit phospholipase D
(23). We achieved the best results with these inhibitors when added
once, after the cells had been grown at 40 °C for 24 h. Further
additions of the drugs resulted in increased cell death (data not
shown). Fig. 6 shows that the addition of
these phospholipase inhibitors rescued the PEMT2-expressing MT58 cells
to various degrees with the most effective being 300 nM
MAFP, the calcium-independent phospholipase A2 inhibitor (22). The addition of MAFP, at 24 h, rescued PEMT2-expressing MT58
cells by about 50% at 48 and 72 h. Fig.
7 shows that MAFP treatment also
increased the cellular level of PC at 48 and 72 h. Although PC
levels were not restored to the same levels as wild-type cells grown at
40 °C (94 ± 6 and 113 ± 6 nmol/mg protein at 48 and
72 h, respectively), the levels were comparable to PC levels in
PEMT2-expressing MT58 cells levels grown at 33 °C (75 ± 6 and
69 ± 7 nmol/mg protein at 48 and 72 h, respectively).
Taken together, the data suggest that the reason PEMT2 does not rescue
the MT58 cell line at 40 °C is that these cells simply do not
produce enough PC for cellular replication at 40 °C. When the cells
are shifted to 40 °C, PC degradation by enzymes such as
phospholipases is greater than PC synthesis in the mutant and PEMT2-expressing MT58 cells. Hence, there is not enough PC produced for
cellular replication and the cells undergo apoptosis (9).
We have investigated why expression of PEMT2 does not rescue the
CT mutant CHO cell line, MT58 at 40 °C. The most obvious hypotheses
at the time were: 1) that PC derived from the PEMT-pathway was not
nuclear whereas PC synthesis from CDP-choline was, and 2) that the two
pathways were producing different molecular species of PC. We found
that neither of these was the case.
First, we found that there was no difference in the synthesis of
nuclear of PC between PEMT2-expressing MT58 cells and CT-expressing MT58 cells. This suggested that differences in the subcellular localization of PC did not account for why PEMT2 could not rescue the
mutant cell line. This conclusion was supported by the recent discovery
of a second isoform of CT, CT- Second, we found that alkyl-LPC as well as acyl-LPC rescued the MT58
cells, indicating that the sn-1 linkage in PC was not critical for cellular replication and homeostasis. DeLong and co-workers (26) found, using McA-RH7777 cells transfected with PEMT2
cDNA, that the molecular species of PEMT-derived PC was more
diverse and contained a higher percentage of arachidonate than did PC
derived from the CDP-choline pathway. However, we found no major
differences in the fatty acid composition of PC, by gas chromatography
analysis, between CT-expressing and PEMT2-expressing MT58 cells grown
at 40 °C after 48 or 72 h. We selected these time points for
our study as these are the times when the PEMT2-expressing MT58 cells
failed to proliferate. These apparently conflicting results may be due
to lipid remodeling occurring in the McA-RH7777 cells.
We now provide evidence that expression of PEMT2 does not rescue the
MT58 cells simply because not enough PC is produced after 24 h.
Four lines of evidence support this conclusion. 1) Medium containing
delipidated serum failed to rescue PEMT2-expressing MT58 cells at
40 °C, whereas medium containing lipids did. 2) PC levels did not
increase in PEMT2-expressing MT58 cells at 40 °C, whereas PC levels
increased in wild-type and CT-expressing MT58 cells. 3) Increased
expression of PEMT2 by transient transfection enhanced the growth of
PEMT2-expressing MT58 cells at 40 °C. 4) Inhibition of
phospholipases allowed these cells to replicate at 40 °C.
We found that, at 40 °C, the PC content of wild-type and
CT-expressing MT58 cells increased (Fig. 3A). Although this
was not totally unexpected, as the laws of thermodynamics would dictate that enzymatic rates increase due to the change in temperature from
33 °C to 40 °C, we did not anticipate such an increase in PC
mass. Several groups have reported that PC mass does not increase even
when CT activity increases (16, 27-29). Excess PC appears to be
degraded by a lipid remodeling phospholipase A2 (16,
28-30). Walkey and co-workers (27) were the first to show that
overexpression of CT activity by transfection of the CT cDNA into
COS cells increases the synthesis of PC, but not the mass of PC. This
phenomenon was also observed in the CT-expressing MT58 cells when grown
at 33 °C (7). The wild-type and CT-expressing MT58 cells do not
exhibit an altered morphology at 40 °C, as one might expect with
such an increase in PC mass.
In contrast, PC levels did not increase in the PEMT2-expressing MT58
cells (Fig. 3A). It was first reported that PC levels, based
on percentage of total phospholipids, are maintained in the
PEMT2-expressing MT58 cells (7) and that on that basis the level of PC
in the PEMT-expressing MT58 cells is the same as the wild-type cells
grown at 40 °C for 24 h. The current analysis, however, is
based upon mass PC per milligram of protein and analyzes changes after
24 h. We were especially interested in time points after 24 h, when the difference between the growth curves of the PEMT2-expressing MT58 cells and wild-type cells was more pronounced. We
found that the difference in PC levels between the PEMT2-expressing MT58 cells and wild-type or CT-expressing MT58 cells is also more pronounced after growth at 40 °C for 48 and 72 h (Fig.
3A). We considered the possibility that PEMT2 may not be
produced or active in PEMT2-expressing MT58 cells grown at 40 °C for
over 24 h. However, active PEMT2 was present in cells grown at
40 °C for at least 72 h (Fig. 3B). In fact, the
level of PEMT activity at 40 °C was higher than the activity at
33 °C (Fig. 3B), consistent with thermodynamic predictions.
We found that products of further PC metabolism, such as TG and LPC,
accumulated in PEMT2-expressing MT58 cells compared with wild-type
cells grown at 40 °C (Fig. 4 and this report). Recently, Jackowski
and co-workers have shown that TG levels increase in the MT58 cells due
to the diversion of newly synthesized diacylglycerol to the TG pool
(17). As shown in Fig. 4, we too see these results. However, some of
the TG accumulated at 40 °C may also result from PC degradation. We
found that the accumulation of TG was greater in the PEMT2-expressing
MT58 cells compared with MT58 cells. This increased accumulation in the
PEMT2-expressing MT58 cells is probably due to increased synthesis of
PC and its subsequent catabolism, compared with the MT58 cells.
Enhanced catabolism of PC is indicated, since LPC levels were higher in
PEMT2-expressing MT58 cells compared with CT-expressing MT58 cells.
Based on the above results, we postulate that at 40 °C, the rate of
PC synthesis is greater than or equal to the rate of PC degradation in
wild-type and CT-expressing MT58 cells and that in MT58 and
PEMT2-expressing MT58 cells grown at 40 °C the rate of PC synthesis
is less than or equal to the rate of PC degradation. If this hypothesis
were true, we should therefore be able to rescue, at least partially,
the PEMT2-expressing MT58 cells if the equilibrium between synthesis
and degradation was shifted in the direction of synthesis. By
transfecting the PEMT2-expressing MT58 cells with additional amounts of
PEMT2 cDNA, the number of viable cells increased at 40 °C,
presumably due to increased synthesis of PC (Fig. 5). In our attempts
to modulate the rate of PC degradation, we showed that several
phospholipase inhibitors increased the growth of PEMT-expressing MT58
cells to varying degrees (Fig. 6). As expected, based upon the reports
of others (16, 28-30), which implicate a role for the
calcium-independent phospholipase A2 in lipid remodeling,
the most effective restoration of growth occurred with inhibitors of
calcium-independent phospholipase A2: MAFP (22) and
manoalide (20, 21). Cell growth was not completely rescued at 40 °C,
as high levels of MAFP were cytotoxic, even at 33 °C. Treatment with
MAFP also increased PC levels in the PEMT-expressing MT58 cells grown
at 40 °C (Fig. 7). These inhibitors did not partially rescue MT58
cells, as we would expect since these cells contain no active
PC-producing pathway at the restrictive temperature.
Taken together, we have shown that the reason why PEMT2 does not rescue
growth of the MT58 cells at 40 °C is that insufficient PC is
produced after 24 h. At 40 °C, the rate of PC degradation is
greater than the rate of PC synthesis in the MT58 and PEMT2-expressing MT58 cells. It remains to be seen if PEMT-derived PC has a specialized role in the whole animal, particularly the liver. The generation of the
PEMT null mouse (31) will help to elucidate further why the liver has
maintained two pathways for PC production. Studies to address this
question are currently under way and should prove to increase our
knowledge on the roles of PC. Nonetheless, the data shown here suggest
that in cell culture, PEMT-derived PC can substitute for PC made by the
CDP-choline pathway in maintaining cellular homeostasis and further
indicates the importance of PC for cellular replication.
We thank Susanne Lingrell for excellent
technical assistance on this project and Dr. Jean Vance for helpful comments.
*
This work was supported in part by the Medical Research
Council of Canada.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Medical scientist of the Alberta Heritage Foundation for Medical
Research. To whom correspondence should be addressed. Fax: 780-492-3383; E-mail: dennis.vance@ualberta.ca.
Published, JBC Papers in Press, May 4, 2000, DOI 10.1074/jbc.M003539200
The abbreviations used are:
PC, phosphatidylcholine;
CT, CTP:phosphocholine cytidylyltransferase;
PE, phosphatidylethanolamine;
CHO, Chinese hamster ovary;
PEMT, phosphatidylethanolamine N-methyltransferase;
LPC, lysophosphatidylcholine;
MAFP, methyl arachidonyl fluorophosphonate;
TG, triacylglycerol;
TLC, thin layer chromatography;
FBS, fetal bovine
serum;
PBS, phosphate-buffered saline;
DOTAP, dioleoyl-trimethylammonium propane.
Why Expression of Phosphatidylethanolamine
N-Methyltransferase Does Not Rescue Chinese Hamster Ovary
Cells That Have an Impaired CDP-Choline Pathway*
and
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ABSTRACT
INTRODUCTION
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rescued the cells at 40 °C, whereas expression of phosphatidylethanolamine
N-methyltransferase-2 (PEMT2) did not, even though PC
levels appeared to be maintained at wild-type levels after 24 h at
the restrictive temperature. We report that the failure of PEMT2 to
rescue the MT58 cell line is due to inadequate long term PC synthesis.
We found that changing the medium every 24 h rescued the
PEMT2-expressing MT58 cells grown at 40 °C. This was due to the
uptake and utilization of lipids in the serum. At 40 °C, PC levels
in the wild-type CHO cells and CT-expressing MT58 cells increased over
time whereas PC levels did not change in both the MT58 and
PEMT2-expressing MT58 cell lines. Further investigation found that both
the PEMT2-expressing MT58 and MT58 cell lines accumulated
triacylglycerol at 40 °C. Pulse-chase experiments indicated that
lyso-PC accumulated to a higher degree at 40 °C in the
PEMT2-expressing MT58 cells compared with CT-expressing MT58 cells.
Transfection of the PEMT-expressing MT58 cells with additional PEMT2
cDNA partially rescued the growth of these cells at 40 °C.
Inhibition of PC degradation, by inhibitors of phospholipases, also
stimulated PEMT-expressing MT58 cell growth at 40 °C. Best results
were observed using a calcium-independent phospholipase A2
inhibitor, methyl arachidonyl fluorophosphonate. This inhibitor also
increased PC mass in the PEMT2-expressing MT58 cells. When the cells
are shifted to 40 °C, PC degradation by enzymes such as
phospholipases is greater than PC synthesis in the mutant
PEMT2-expressing MT58 cells. Taken together, these results indicate
that PEMT2 expression fails to rescue the mutant cell line at 40 °C
because it does not maintain PC levels required for cellular replication.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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rescued the cells whereas cDNA encoding an isoform
of PEMT, PEMT2, did not. This occurred even though PC levels, based
upon percentage of total phospholipid, appeared to be restored (7).
These cells provide an excellent system in which to analyze the role of
both CDP-choline- and PEMT-derived PC in cellular replication and
investigate why PEMT-derived PC does not rescue the mutant cell line.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Changing medium rescues PEMT2-expressing MT58
cells at 40 °C. PEMT-expressing MT58 cells were plated on 60-mm
dishes with 4 ml of medium at a density of 2.5 × 105
cells/dish. The cells were then incubated at either 33 °C or
40 °C. At various times, the medium was either removed and replaced
with fresh medium (diamonds) or the medium was not changed
(circles 40 °C, triangles 33 °C). At the indicated
times, the cells were harvested using trypsin and viable cells were
counted. Shown are the means ± S.E. of six individual
experiments.
View larger version (17K):
[in a new window]
Fig. 2.
Medium containing delipidated serum does not
rescue PEMT2-expressing MT58 cells at 40 °C. PEMT2-expressing
MT58 cells were plated on 60-mm dishes with 4 ml of medium at a density
of 2.5 × 105 cells/dish. The cells were then
incubated at either 40 °C (panel A) or
33 °C (panel B). At the indicated times, some
dishes of cells were harvested using trypsin and viable cells were
counted. To the remaining dishes the medium was either not changed
(circles), removed and replaced with fresh medium containing
FBS (+Medium, diamonds), or removed and replaced
with fresh medium containing delipidated FBS (+Delip.
Serum, triangles). Shown are the means ± S.E. of six individual experiments for cells grown at 40 °C,
33 °C, and 40 °C plus fresh medium containing FBS, and three
individual experiments for cells grown in medium containing delipidated
serum.
View larger version (36K):
[in a new window]
Fig. 3.
PC levels fail to increase in
PEMT2-expressing MT58 cells grown at 40 °C. Panel
A, PC mass. CHO cells were grown on 100-mm dishes; when the
cells were about 30% confluent, they were shifted to 40 °C (0 h).
At the indicated times, the cells were harvested and subjected to lipid
extraction followed by TLC to separate PC. Phosphorous content was
determined as described under "Experimental Procedures." Shown are
the means ± S.E. of three individual experiments.
Panel B, [3H]methionine
incorporation into PC. PEMT2-expressing MT58 cells were grown on 100-mm
dishes. When the cells were about 30% confluent, half of the cells
were shifted to 40 °C and the remaining cells were grown at
33 °C. At the indicated times, 5 µCi of
[3H]methionine was added to the medium for 2 h. The
cells were then washed, harvested, and subjected to lipid extraction
followed by TLC to separate the radiolabeled PC. The PC was visualized
by iodine vapor and scraped, and radioactivity was measured by liquid
scintillation counting. Shown are the means of two individual
experiments.
View larger version (33K):
[in a new window]
Fig. 4.
Accumulation of TG in mutant and
PEMT2-expressing MT58 cells at 40 °C. CHO cells were grown on
100-mm dishes. When the cells were about 30% confluent (0 h), some
were kept at 33 °C (open symbols) and others
were incubated at 40 °C (closed symbols). At
the indicated times, the cells were harvested and subjected to lipid
extraction and TLC. TG was visualized by iodine vapor and scraped. The
mass of TG was determined by the hydroxlamine method (14). Shown are
the mean ± S.E. of three individual experiments.
View larger version (20K):
[in a new window]
Fig. 5.
Transient transfection with PEMT2 cDNA
partially rescues PEMT-expressing MT58 cells at 40 °C.
PEMT2-expressing MT58 cells were plated on 60-mm dishes with 4 ml of
medium at a density of 2.5 × 105 cells/dish and
incubated at 33 °C overnight. The medium was removed and replaced
with serum-free medium. Next, the cells were transfected with 5 µg/dish of either PEMT2 cDNA (Transfected) or empty
vector (Control) as described under "Experimental
Procedures." After 4 h, the medium was removed and replaced with
fresh culture medium and the plates were shifted to 40 °C. At the
indicated times, the cells were harvested using trypsin and viable
cells were counted. Shown are the means ± S.E. of three
individual experiments; some error bars are too small to be
observed.
View larger version (25K):
[in a new window]
Fig. 6.
Phospholipase inhibitors partially rescue
growth of PEMT-expressing MT58 cells at 40 °C. PEMT2-expressing
MT58 cells were plated on 60-mm dishes with 4 ml of medium at a density
of 2.5 × 105 cells/dish and grown at 40 °C. After
24 h, 10 µM D609 (closed
diamonds), 100 nM manoalide (closed
circles), 300 nM MAFP (closed
triangles), 5 µg/ml L6598989 (closed
inverted triangles) or dimethyl sulfoxide as a
vehicle control (No Treatment; open
squares) was added to the dishes. At the indicated times,
the cells were harvested by trypsin and viable cells were counted.
Shown are the means ± S.E. of five individual experiments.
View larger version (94K):
[in a new window]
Fig. 7.
MAFP treatment maintains PC levels in
PEMT2-expressing MT58 cells at 40 °C. PEMT2-expressing MT58
cells were grown on 100-mm dishes at 40 °C. After 24 h, 300 nM MAFP was added to some dishes. At the indicated times,
the cells were harvested and subjected to lipid extraction followed by
TLC to separate PC. Phosphorous content was determined as described
under "Experimental Procedures." Shown are the means ± S.E.
of three individual experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (24).
CT-1 does not contain the nuclear targeting sequence and
has been found to associate with the endoplasmic reticulum.
Transfection with the cDNA for CT-1 rescues the MT58
cell line at 40 °C (25).
ACKNOWLEDGEMENTS
FOOTNOTES
Postdoctoral fellow of the Alberta Heritage Foundation for Medical Research.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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