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J Biol Chem, Vol. 275, Issue 12, 9078-9084, March 24, 2000
From the The de novo pathway of sphingolipid
synthesis has been identified recently as a novel means of generating
ceramide during apoptosis. Furthermore, it has been suggested that the
activation of dihydroceramide synthase is responsible for increased
ceramide production through this pathway. In this study, accumulation
of ceramide mass in Molt-4 human leukemia cells by the chemotherapy agent etoposide was found to occur primarily due to activation of the
de novo pathway. However, when the cells were labeled with a substrate for dihydroceramide synthase in the presence of etoposide, there was no corresponding increase in labeled ceramide. Further investigation using a labeled substrate for serine
palmitoyltransferase, the rate-limiting enzyme in the pathway, resulted
in an accumulation of label in ceramide upon etoposide treatment. This
result suggests that the activation of serine palmitoyltransferase is
the event responsible for increased ceramide generation during de
novo synthesis initiated by etoposide. Importantly, the ceramide
generated from de novo synthesis appears to have a distinct
function from that induced by sphingomyelinase action in that it is not
involved in caspase-induced poly (ADP-ribose)polymerase proteolysis but does play a role in disrupting membrane integrity in this model system.
These results implicate serine palmitoyltransferase as the enzyme
controlling de novo ceramide synthesis during apoptosis and
begin to define a unique function of ceramide generated from this pathway.
It is increasingly apparent that sphingolipids, and in particular
ceramide, are important mediators in regulating the response to stress
of a cell. The agents that induce ceramide generation include
physiological factors, such as tumor necrosis factor and the Fas
ligand, as well as therapeutic agents, such as chemotherapy drugs and
radiation. Many of these agents induce ceramide generation via the
hydrolysis of sphingomyelin by the activation of one or more
sphingomyelinases. Additional studies, however, have begun to implicate
ceramide generated from the de novo pathway of sphingolipid synthesis as having a signaling function (1-8).
Studies of de novo sphingolipid biosynthesis have been
advanced by the realization that a class of fungal metabolites known as
fumonisins share structural similarities with the sphingoid backbone.
During investigation of the effects of fumonisin on sphingolipid
metabolism in hepatocytes, it was observed that the synthesis of
complex sphingolipids was significantly inhibited. It was also
determined that the primary site of action of fumonisin was
dihydroceramide synthase (9), an enzyme in the de novo pathway that catalyzes the N-acylation of sphinganine to
produce dihydroceramide.
Because ceramide has been implicated as a regulatory molecule in
apoptosis, more recent studies have used fumonisins to investigate the
potential role of ceramide from the de novo pathway in this process. These studies have demonstrated that fumonisin is able to
attenuate apoptosis induced by daunorubicin, camptothecin, tumor
necrosis factor- In this study, we have used the chemotherapy agent etoposide to
activate de novo ceramide synthesis in Molt-4 human leukemia cells. In preliminary experiments, we were unable to find any evidence
for activation of dihydroceramide synthase, and we became interested in
determining the regulatory point in de novo ceramide synthesis under apoptotic conditions with etoposide. Using intact cell
radiolabeling techniques and cell-free enzyme assays, we determined
that serine palmitoyltransferase, the initial and rate-limiting enzyme
in the pathway, is activated during apoptosis and governs the
production of ceramide.
We were also interested in elucidating a regulatory function for
ceramide generated de novo in apoptosis, and our present studies demonstrate that it has its predominant effects on
membrane-related events in apoptosis. Importantly, unlike short-chain
ceramide or ceramide generated from sphingomyelinase action, this
ceramide is dissociated from caspase activation. This study provides
the first evidence that serine palmitoyltransferase is a regulated enzyme during apoptosis and provides further evidence that ceramide generated de novo functions as a regulatory molecule in
mediating membrane-related apoptotic events.
Materials--
Ceramide standards were derived from
phospholipase C hydrolysis of brain sphingomyelin and were purchased
from Avanti Polar Lipids (Alabaster, AL). [ Cell Culture--
Molt-4 cells were obtained from ATCC
(Manassas, VA) and maintained at 37 °C and 5% CO2 in
RPMI medium with 10% fetal bovine serum. Cells were subcultured prior
to reaching a density of 2 × 106/ml.
Ceramide Mass Measurements--
Molt-4 cells were seeded at
5 × 105/ml (3 mls/well in a 6-well plate). After
treatments, ceramide mass was determined using the diglyceride kinase
assay as described previously (11). Briefly, 5 × 105
cells were lysed in a mixture of chloroform and methanol (1:2), and
lipids were extracted according to Bligh and Dyer (12). Lipids were
solubilized in dioleoylphosphatidylglycerol/ Lipid Phosphate Determination--
Lipid phosphate was measured
by acid hydrolysis of a Bligh-Dyer extract from 106 Molt-4
cells. The chloroform phase of the extract was evaporated under
N2, and the lipids were incubated at 150 °C for 6 h
in 0.6 ml of 10 N H2SO4/70%
HClO4/H2O (9:1:40). After acid hydrolysis, 0.6 ml of H2O, 0.5 ml of 0.9% ammonium molybdate, and 0.2 ml
of 9.0% ascorbic acid were added and incubated for 30 min at 45 °C. Inorganic phosphate was detected by absorbance at 820 nm and quantified based upon a standard curve of K2HPO4.
Radiolabeling of Cells--
Molt-4 cells were suspended at
5 × 105/ml in RPMI medium with 2% fetal calf serum.
Five ml of this suspension were added to each flask, and cells were
labeled with either 0.1 µCi of [3H]sphinganine (0.045 µCi/nmol) or 5 µCi of [3H]palmitate (43 Ci/mmol).
Concurrent with the addition of radionuclide, etoposide and inhibitors
of de novo synthesis were added. After 6 h, cells were
harvested by centrifugation at 4 °C. Lipids were then extracted
according to Bligh and Dyer (12). Lipid extracts from cells labeled
with [3H]sphinganine were immediately run on TLC. Lipids
extracted from [3H]palmitate-labeled cells were first
submitted to mild alkaline hydrolysis prior to separation by TLC. The
solvent system employed was chloroform/methanol/2 N
NH4OH, 4:1:0.1. After chromatography, a ceramide standard
(RF = 0.77) was identified by the use of iodine
vapors. The TLC plates were then sprayed lightly with
En3hance, and radioactivity was visualized by
autoradiography after 48 h at -80 °C. The radioactive spot
migrating with the same RF as the ceramide standard
was scraped from the plate and quantified by liquid scintillation spectrometry.
RT-PCR of Human Serine Palmitoyltransferase Subunits--
Total
cellular RNA was isolated from 2.5 × 106 human Molt-4
cells at each time point that had been treated with 10 µM
etoposide or left untreated at 37 °C. RNA was subjected to RT-PCR on
a Roche Molecular Biochemicals LightCycler as follows: a 20-µl
reaction containing 200 ng of RNA, 0.25 µM each primer, 5 mM MgCl2, 1× LightCycler RT-PCR reaction
mixture SYBR Green, and 1× LightCycler RT-PCR enzyme mixture was
amplified in a LightCycler capillary. The sample was reverse
transcribed at 55 °C for 10 min and heated to 95 °C for 30 s. The resulting cDNA was amplified for 45 cycles by denaturation
at 95 °C for 1 s, primer annealing at 55 °C for 10 s,
and elongation at 72 °C for 20 s. One fluorescence reading was
taken in each cycle following the elongation step. The primers used were as follows: human b-actin, 5'-TCCTGTGGCATCCACGAAACT-3' and
5'-GAAGCATTTGCGGTGGACGAT-3'; human serine palmitoyltransferase subunit
1 (hLCB1), 5'-AGGAGTCACTGAACACTATG-3' and 5'-AGCTCTCTCCAGTTCTTCCT-3'; and human serine palmitoyltransferase subunit 2 (hLCB2),
5'-GTGGATGTTATGATGGGAACG-3' and 5'-CATACGTCGTCTCGTCAAAG-3'. The
GenBankTM accession numbers for these genes are as follows:
b-actin, X00351; hLCB1, Y08685; and hLCB2, Y08686.
Enzyme Assays--
Serine palmitoyltransferase was assayed as
described previously (13). Briefly, enzyme activity in 100 µg of
microsomal membranes was determined in 100 mM Hepes (pH
8.3), 5 mM dithiothreitol, 2.5 mM EDTA (pH
7.4), and 50 µM pyridoxal 5'-phosphate. The reaction was
initiated by the addition of 200 µM palmitoyl CoA and 2 µCi of L-[3H]serine, with a final serine
concentration of 1 mM. Reactions were incubated for 10 min
at 37 °C prior to termination with 0.2 ml of 0.5 N NaOH. Organic
soluble counts were extracted as described previously and quantified by
liquid scintillation counting (13).
Dihydroceramide synthase was assayed according to Bose et
al. (1). Briefly, the dihydroceramide synthase activity in 50 µg
of microsomal membranes was determined in 20 mM Hepes (pH
7.4), 2 mM MgCl2, 20 µM fatty
acid-free bovine serum albumin, and 20 µM sphinganine.
The reaction was initiated by the addition of 70 µM
palmitoyl CoA containing 0.5 µCi of [3H]palmitoyl CoA
and allowed to incubate for 30 min at 37 °C. Lipids were extracted
by the method of Bligh and Dyer (12) and resolved by thin layer
chromatography in a solvent system of chloroform/methanol/2 N NH4OH (4:1.0:0.1). Radioactivity comigrating
with a ceramide standard was scraped from the plate and quantified by
liquid scintillation counting.
Western Blotting--
PARP proteolysis was determined as
described previously (14). Approximately 5 × 105
cells were lysed by boiling in Laemmli buffer, and the lysate was
electrophoresed on a 6% polyacrylamide gel. After transferring proteins to nitrocellulose, the blot was incubated for 1 h with rabbit anti-PARP (1:2000), and detection was performed by enhanced chemilumenescence using peroxidase-conjugated goat anti-rabbit (1:5000).
Cell Viability Assays--
Molt-4 cells were suspended at 5 × 105/ml in RPMI medium with 2% fetal calf serum. Two ml
of the cell suspension were aliquoted into the wells of a 12-well
plate. Cells were treated simultaneously with etoposide and fumonisin.
After 9 or 24 h, 0.5 ml of cells was removed from the well,
harvested by centrifugation, and suspended in 0.2 ml of
phosphate-buffered saline. Following the addition of 0.2 ml of 0.4%
trypan blue solution, cells were examined with a light microscope for
trypan blue staining.
Induction of de Novo Synthesis of Ceramide by Etoposide--
The
chemotherapy agent etoposide initiates an apoptotic response by
inhibiting topoisomerase II, resulting in single-stranded DNA breaks
(15). Through unknown mechanisms, this insult by etoposide results in
the release of cytochrome c from the mitochondria (16),
activation of caspases (17), DNA fragmentation (18), and subsequent
cell death (reviewed in Ref. 19). Because ceramide has been shown to be
an inducer of these events (20-22), we were interested in determining
whether etoposide elevated ceramide levels and, if so, in determining
the origin of ceramide. As demonstrated in Fig.
1, treatment with etoposide resulted in a
nearly 3-fold elevation of ceramide after 6 h. In the presence of
fumonisin, an inhibitor of de novo ceramide synthesis,
etoposide-induced ceramide accumulation was decreased by nearly 75%
after 6 h.
Radiolabeling of Cells with a Serine Palmitoyltransferase Substrate
but Not a Dihydroceramide Synthase Substrate Results in Increased
Incorporation of Radiolabel into Ceramide upon Etoposide
Treatment--
To determine whether induction of de novo
ceramide generation was occurring by activation of dihydroceramide
synthase as previously reported using daunorubicin (1), cells were
radiolabeled with [3H]sphinganine, the substrate for this
enzyme (Fig. 2), and simultaneously treated with 10 µM etoposide. Using this method and
measuring [3H]ceramide over a 9-h time course with
etoposide, no increased incorporation of label into ceramide was
observed (Fig. 3A). This result suggests that neither dihydroceramide synthase nor the subsequent enzyme in de novo synthesis, dihydroceramide
desaturase, is responsible for increased synthesis of ceramide.
In a similar experiment using a 6 h time period of etoposide
treatment in the absence or presence of fumonisin, the incorporation of
[3H]sphinganine into [3H]ceramide was
significantly inhibited (Fig. 3B), consistent with the
reported inhibition of dihydroceramide synthase by fumonisin (23).
The lack of an increase in de novo ceramide synthesis using
a dihydroceramide synthase substrate indicated a prior step in the
pathway as the site of activation. Cells were therefore radiolabeled with [3H]palmitate, a precursor to the serine
palmitoyltransferase substrate palmitoyl CoA, and simultaneously
treated with etoposide. An increase of over 3-fold in
[3H]ceramide was observed by 9 h (Fig.
4A), suggesting that serine palmitoyltransferase, the rate-limiting enzyme in de novo
synthesis of sphingolipids, is activated during de novo
synthesis of ceramide under apoptotic stress. Moreover, both fumonisin
and cycloserine, an inhibitor of serine palmitoyltransferase (24),
inhibited the incorporation of [3H]palmitate into
[3H]ceramide during a 6 h time period of etoposide
treatment (Fig. 4B).
Regulation of Serine Palmitoyltransferase
Activity--
Previously, it had been demonstrated that during
irradiation of keratinocytes, RNA levels of LCB2, a serine
palmitoyltransferase subunit, were up-regulated (25). Therefore, in
order to understand the regulation of serine palmitoyltransferase
activity during etoposide-induced apoptosis, we initially determined
mRNA levels of the LCB1 and LCB2 subunits of serine
palmitoyltransferase by RT-PCR. No increases in the message level of
either subunit were observed. In contrast, the serine
palmitoyltransferase RNA was degraded by 6 h of etoposide
treatment (Table I). Because
up-regulation of RNA was not responsible for increased enzyme activity,
in vitro enzyme assays for serine palmitoyltransferase were
conducted using microsomes from either control- or etoposide-treated
cells. Over a 6-h course of treatment, serine palmitoyltransferase
activity was elevated early (by 0.5 h) and sustained throughout
the treatment (Fig. 5). These results
rule out etoposide-induced activation of serine palmitoyltransferase by
up-regulation of RNA and suggest activation by covalent modification or
allosteric regulation of the enzyme.
Dissociation of de Novo Synthesized Ceramide from Caspase
Activation--
Because we and others have previously demonstrated
that short-chain ceramide induces PARP proteolysis by caspase
activation (21, 26) and because etoposide had previously been
demonstrated to cause PARP proteolysis (18), we were interested in
determining whether the de novo generation of ceramide by
etoposide was also instrumental in this caspase-mediated event. In the
presence of fumonisin, etoposide-induced de novo ceramide
generation returned to basal levels yet PARP proteolysis was unaffected
(Figs. 4B and 6). This result
demonstrates that de novo ceramide generation is not
necessary for the activation of a PARP-cleaving caspase(s) in this cell
system. In addition, we have also examined the effect of fumonisin on
lamin B cleavage by etoposide, reportedly occurring by caspase-6 (27),
and found no inhibition (data not shown). Additional studies using the
chromogenic caspase substrates YVAD-pNA, DEVD-pNA, and IETD-pNA were
also conducted. These peptides function as substrates for group I,
group II, and group III caspases, respectively (28). Whereas etoposide
induced both DEVD-pNA and IETD-pNA peptidase activity, fumonisin was
without an inhibitory effect (data not shown). These results are
consistent with prior studies demonstrating etoposide activation of
caspase-3 (of which DEVD-pNA is a substrate), and caspase-6 (of which
IETD-pNA is a substrate) in human leukemia cells (29). Furthermore,
they provide evidence for the dissociation of de novo
generated ceramide from caspase activation and serve to distinguish its
function from both ceramide generated from sphingomyelinases and from
exogenous short-chain ceramides, both of which have been implicated in
activation of caspases (21, 26, 30).
Effect of Fumonisin on Cell Death--
Finally, we were interested
in determining whether the inhibition of de novo generated
ceramide by fumonisin protected cells from death. Because noticeable
cell death did not occur during the first 6 h of treatment with
etoposide, we assayed cell death at 9 or 24 h after treatment and
observed nearly 15 and 75% cell death, respectively, as determined by
trypan blue staining (Fig. 7). When the
cells were treated during this period with either fumonisin or
zVAD-fmk, a pancaspase inhibitor, cell death was approximately 7%
after 9 h and 30% after 24 h. Interestingly, when fumonisin
and zVAD-fmk were added together, near complete protection from cell
death was observed. These results suggest that ceramide from de
novo synthesis and caspases contribute to independent pathways of
death during etoposide-induced apoptosis.
The results from this study provide the first evidence that the
initial and rate-limiting enzyme in the de novo pathway of sphingolipid biosynthesis, serine palmitoyltransferase, is activated during apoptosis. They also provide further support for a regulatory role in apoptosis of ceramide generated from the de novo pathway.
Currently, very little is known about the function of serine
palmitoyltransferase other than its role in sphingolipid synthesis for
housekeeping functions, but studies in Saccharomyces
cerevisiae have begun to define a role for the enzyme in stress
response signaling. A yeast strain lacking serine palmitoyltransferase activity has been identified that is unable to grow unless supplemented with sphingoid bases (31). Suppressors of this mutation have also been
isolated that are able to grow at ambient temperature but are unable to
survive hyperosmolar or heat stress (32). The suppressor strains could
be rescued, however, either by transfection with the serine
palmitoyltransferase or by supplying exogenous sphingoid bases (33),
thus implicating serine palmitoyltransferase in both heat and osmotic
stress responses.
In mammalian cells, it has been demonstrated that sphingoid bases are
capable of down-regulating serine palmitoyltransferase (34) and that
the activity of the enzyme progressively increases during the
differentiation process of neuronal cells in culture (35). Moreover, a
recent report indicated that 48 h after UV irradiation of
keratinocytes, mRNA levels for the LCB2 subunit of serine
palmitoyltransferase were up-regulated 1.7-fold and that this
corresponded to a 1.5-fold increase in enzyme activity (25).
Our results suggest that caution should be used in interpreting data
obtained with the dihydroceramide synthase inhibitor, fumonisin. An
inhibition of ceramide accumulation with fumonisin, although
implicating de novo synthesis, does not imply that
dihydroceramide synthase is the regulatory enzyme. Such a result would
also be consistent with a regulatory role of any of the preceding
enzymes in the pathway. In two recent studies of phorbol ester- and
daunorubicin-induced apoptosis mediated by de novo ceramide
synthesis, in vitro assays of dihydroceramide synthase
indicated that the Vmax was increased 1.6-1.7-fold after treatment (1, 5). However, in our intact cell
[3H]sphinganine labeling experiment using etoposide or
using an in vitro enzyme assay (data not shown), we found no
evidence that dihydroceramide synthase is activated during de
novo synthesis in response to etoposide.
The enzymes of the de novo pathway (Fig. 2) leading to the
production of ceramide reside on the endoplasmic reticulum (36). In
studies using hepatocytes, it was determined that the specific activity
of serine palmitoyltransferase is considerably less than that of other
enzymes in the pathway, including ketosphinganine reductase and
dihydroceramide synthase. This fact, in conjunction with the
observation of low sphingoid base levels in cells, has led to the
conclusion that serine palmitoyltransferase is the rate-limiting enzyme
in the pathway of sphingolipid biosynthesis (13). Because the
concentrations of reactants for a rate-limiting enzyme are generally
much higher than the products, the activation of such an enzyme would
be expected to shift the reaction toward equilibrium and result in a
deregulation of the pathway. Under the experimental conditions in this
study, activation of serine palmitoyltransferase would therefore have
the consequence of leading to accumulation of an end product of the pathway.
Under normal conditions of de novo sphingolipid synthesis,
ceramide is not typically considered an end product because it serves
as a substrate for sphingomyelin synthase and glucosylceramide synthase, enzymes of complex sphingolipid synthesis. Whereas the enzymes of the de novo pathway (Fig. 2) leading to the
production of ceramide reside on the endoplasmic reticulum (36), the
sphingomyelin and glucosylceramide synthases are located on the Golgi
apparatus and/or plasma membrane (37, 38). Therefore, our data
demonstrating the accumulation of ceramide from de novo
synthesis indicate that either the transport of ceramide to these
locations or the respective synthases may serve as additional
regulatory points in this pathway during conditions of stress.
Additionally, if etoposide induced the accumulation of de
novo ceramide by inhibiting one of the enzymes that use ceramide as a substrate, similar accumulations of ceramide should have been
observed with both palmitate and sphinganine as substrates in the
intact cell labeling experiments. The observation that elevated
ceramide was seen only with palmitate as a substrate further supports
the conclusion that ceramide accumulation is primarily due to
activation of serine palmitoyltransferase.
We and others had previously shown that short-chain ceramide can induce
PARP proteolysis and caspase activation (21, 26). It has also been
reported that ceramide produced by the action of sphingomyelinases is
important for caspase activation (30). Our results demonstrating a lack
of involvement of de novo synthesized ceramide in the
activation of caspases suggest that this pool of ceramide may have a
unique signaling or regulatory function. Because the de novo
pathway synthesizes ceramide on the endoplasmic reticulum and because a
signaling pool of ceramide has also been identified at the plasma
membrane (39), it is plausible that these respective pools of ceramide
employ different effector mechanisms.
These studies with etoposide do not exclude the possibility that other
inducers of de novo synthesis and/or other cell types regulate additional enzymes in the de novo pathway. Also,
the results do not exclude the possibility of additional functions for
de novo generated ceramide in the apoptotic response.
However, it is clear that care should be exercised in defining the main sites of biochemical regulation in the de novo pathway using
a combination of enzymatic and labeling studies. Care should also be
exercised in determining what specific aspects of apoptosis are
regulated (or not regulated) by the de novo pathway.
The results from the trypan blue experiment suggest that de
novo ceramide generated during apoptosis exerts a key regulatory function in effecting membrane damage. Furthermore, the additive and
protective effect of fumonisin and a caspase inhibitor on cell death
provide evidence that ceramide from de novo synthesis and
caspases are activating independent pathways in apoptosis and suggest
the model proposed in Fig. 8.
In summary, the results from this study begin to define serine
palmitoyltransferase as an important regulatory step in the de
novo pathway of ceramide synthesis during apoptosis. Moreover, they dissociate this pathway from a role in caspase activation and
implicate it in mediating membrane-related events in apoptosis.
We thank Dr. Ala Bielawska for the
synthesis of [3H]sphinganine.
*
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.
The abbreviations used are:
PARP, poly(ADP-ribose) polymerase;
RT, reverse transcription;
PCR, polymerase
chain reaction.
Serine Palmitoyltransferase Regulates de Novo
Ceramide Generation during Etoposide-induced Apoptosis*
,,
Department of Biochemistry and Molecular
Biology, Medical University of South Carolina,
Charleston, South Carolina 29425, the § R. W. Johnson
Pharmaceutical Research Institute, Raritan, New Jersey 08869, and the ¶ Richard B. Russell Agricultural Research Station,
Athens, Georgia 30604
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and phorbol ester (1, 3, 5, 6). Because fumonisin
inhibits dihydroceramide synthase, it generally has been assumed that
this is the regulatory step in the de novo pathway during apoptosis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP
(3000 Ci/mmol) and [9,10-3H]palmitic acid (43 Ci/mmol)
were from NEN Life Science Products. [3H]Sphinganine was
synthesized as described previously (10). [9,10-3H]palmitoyl CoA (60 Ci/mmol) and
L-[3H]serine (20 Ci/mmol) were purchased from
American Radiolabeled Chemicals (St. Louis, MO). A rabbit polyclonal
antibody raised to an epitope in the automodification domain of human
poly(ADP-ribose) polymerase
(PARP)1 was a gift of Dr. Guy
Poirier of Laval University (Ste Foy, Quebec, Canada).
-octylglucoside mixed
micelles for 30 min at 37 °C. Three µg of membranes from E. coli strain N4830/pJW10 overexpressing diglyceride kinase
were added, and the reaction was initiated by the addition of 3 µCi of [-32P]ATP (3000 Ci/mmol) in the presence of 1 mM carrier ATP. After 30 min at room temperature, the
reaction was quenched with chloroform/methanol (1:2) and the lipids
extracted by the Bligh-Dyer method. After evaporation of the chloroform
phase under N2, the lipid pellet was suspended in 40 µl
of chloroform/methanol (4:1) and spotted on silica gel 60 TLC plates.
The lipids were chromatographed in a solvent system of
chloroform/acetone/methanol/acetic acid/water (10:4:3:2:1). After
autoradiography, the location of ceramide phosphate was determined
based upon the RF (0.48) of phosphorylated ceramide
standards. Radioactivity was quantified by liquid scintillation
spectrometry. Ceramide mass was then determined using the slope of the
standard curve and normalized to lipid phosphate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Time course for the induction of de
novo synthesis by etoposide. Molt-4 cells were treated
for 0-6 h with 10 µM etoposide in the presence
(solid squares) or absence (open squares) of 50 µM fumonisin. At the indicated times, the cells were
harvested, and ceramide mass was quantified using the diglyceride
kinase assay and normalized to lipid phosphate. Error bars
represent the S.D. of triplicate samples.
View larger version (13K):
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Fig. 2.
The de novo pathway of
ceramide biosynthesis.
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Fig. 3.
Effect of etoposide on the incorporation of
[3H]sphinganine into [3H]ceramide.
A, Molt-4 cells were treated simultaneously with
[3H]sphinganine and 10 µM etoposide for
1-9 h. At the indicated time points, cells were harvested as described
under "Experimental Procedures," and [3H]ceramide was
quantified. Each column represents the percent of
[3H]ceramide relative to the ceramide level in a control
at that time point. Error bars represent the S.D. of
triplicate samples. B, simultaneous with the addition of
[3H]sphinganine, Molt-4 cells were treated with 10 µM etoposide (lane 2) or 50 µM
fumonisin (lane 3) alone or with etoposide and fumonisin
(lane 4). After 6 h, the cells were processed as
described under "Experimental Procedures," and
3H-labeled lipids were analyzed by TLC and autoradiography.
Lane 1, vehicle-treated control.
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Fig. 4.
Effect of etoposide on the incorporation of
[3H]palmitate into [3H]ceramide.
A, Molt-4 cells were treated simultaneously with
[3H]palmitate and 10 µM etoposide for 1-9
h. At the indicated time points, cells were harvested as described
under "Experimental Procedures," and [3H]ceramide was
quantified. Each column represents the percent of
[3H]ceramide relative to the control at that time point.
Error bars represent the S.D. of triplicate samples.
B, [3H]palmitate was added to Molt-4 cells
followed immediately by 10 µM etoposide (lane
2), 50 µM fumonisin (lane 3), or 300 µM cycloserine (lane 5). Alternatively,
etoposide was added with fumonisin (lane 4) or with
cycloserine (lane 6). After 6 h, the cells were
processed as described under "Experimental Procedures," and
3H-labeled lipids were analyzed by TLC and autoradiography.
Lane 1, vehicle-treated control.
RT-PCR of actin, hLCB1, and hLCB2 RNA in etoposide-treated Molt-4 cells
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Fig. 5.
Determination of SPT activity in microsomes
from control- or etoposide-treated Molt-4 cells. Molt-4 cells were
treated from 0-6 h with 10 µM etoposide or
Me2SO control. At the indicated times, cells were
harvested, microsomes were isolated, and SPT activity was determined.
Data are expressed as relative units and represent the mean of
triplicate samples ± S.D.
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Fig. 6.
Induction of PARP proteolysis by etoposide
and the lack of inhibition by fumonisin. Molt-4 cells were treated
for 0-24 h with 10 µM etoposide in the absence or
presence of 50 µM fumonisin. At the indicated times,
cells were harvested and the total cell lysate was electrophoresed on a
6% polyacrylamide gel. PARP proteolysis was determined by Western blot
analysis and the data shown are representative of three independent
experiments.
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Fig. 7.
Trypan blue uptake in etoposide-treated
cells. Molt-4 cells were treated for 9 h (upper
panel) or 24 h (lower panel) with 10 µM etoposide (E) in the absence or presence of
50 µM fumonisin (F) and/or 50 µM
z-Val-Ala-Asp-fluoromethylketone (zVAD). Cells were then
harvested and placed in trypan blue solution for determination of cell
viability. Data shown are representative of three individual
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (14K):
[in a new window]
Fig. 8.
Proposed scheme for etoposide-induced
apoptosis.
ACKNOWLEDGEMENT
FOOTNOTES
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular and Biology, Medical University of South
Carolina, 173 Ashley Ave., Charleston, SC 29425. Fax:
843-953-0843.
ABBREVIATIONS
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DISCUSSION
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H. Wang, B. J. Maurer, C. P. Reynolds, and M. C. Cabot N-(4-Hydroxyphenyl)retinamide Elevates Ceramide in Neuroblastoma Cell Lines by Coordinate Activation of Serine Palmitoyltransferase and Ceramide Synthase Cancer Res., July 1, 2001; 61(13): 5102 - 5105. [Abstract] [Full Text] [PDF]
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 A. Senchenkov, D. A. Litvak, and M. C. Cabot Targeting Ceramide Metabolism--a Strategy for Overcoming Drug Resistance J Natl Cancer Inst, March 7, 2001; 93(5): 347 - 357. [Abstract] [Full Text] [PDF]
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B. J. Maurer, L. Melton, C. Billups, M. C. Cabot, and C. P. Reynolds Synergistic Cytotoxicity in Solid Tumor Cell Lines Between N-(4-Hydroxyphenyl)retinamide and Modulators of Ceramide Metabolism J Natl Cancer Inst, December 6, 2000; 92(23): 1897 - 1909. [Abstract] [Full Text] [PDF]
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A. D. Tepper, S. H. Diks, W. J. van Blitterswijk, and J. Borst Glucosylceramide Synthase Does Not Attenuate the Ceramide Pool Accumulating during Apoptosis Induced by CD95 or Anti-cancer Regimens J. Biol. Chem., October 27, 2000; 275(44): 34810 - 34817. [Abstract] [Full Text] [PDF]
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C. E. Chalfant, B. Ogretmen, S. Galadari, B.-J. Kroesen, B. J. Pettus, and Y. A. Hannun FAS Activation Induces Dephosphorylation of SR Proteins. DEPENDENCE ON THE DE NOVO GENERATION OF CERAMIDE AND ACTIVATION OF PROTEIN PHOSPHATASE 1 J. Biol. Chem., November 21, 2001; 276(48): 44848 - 44855. [Abstract] [Full Text] [PDF]
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