(Received for publication, August 15, 1994; and in revised form, November 8, 1994)
From the
In an attempt to define the basis for sphingolipid regulation of
cell proliferation, we studied the effects of glucosylceramide (GlcCer)
synthase inhibition by
threo1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on NIH
3T3 cells overexpressing insulin-like growth factor-1 (IGF-1)
receptors. PDMP treatment resulted in a time-dependent decrease in
GlcCer levels and an increase in cellular ceramide levels. PDMP
abolished serum and IGF-1-stimulated cell proliferation, as measured by
a reduction in [H]thymidine incorporation,
protein, and DNA levels. However it did not affect IGF-1-mediated early
signaling events, including receptor tyrosine kinase, MAP kinase, and
phosphatidylinositol 3-kinase activities. Two-color flow cytometry with
propidium iodide and 5-bromo-2`-deoxyuridine monophosphate labeling
revealed an arrest of the cell cycle at G
/S and
G
/M transitions in an asynchronous population of cells.
These changes were time dependent, with maximal effects seen by
12-24 h. Removal of PDMP from the cell medium resulted in
reversal of the cell cycle changes, with cells re-entering the S phase.
The cell cycle arrest at the G
/S and G
/M
transitions was confirmed in cells synchronized by pretreatment with
nocodazole, aphidicolin, or hydroxyurea, and released from blockade in
the presence of PDMP. A decrease in the activities of two
cyclin-dependent kinases, p34
kinase and cdk2
kinase, was observed with PDMP treatment. When cell ceramide levels
were increased by N-acetylsphingosine, comparable changes in
the cell cycle distribution were seen. However, sphingomyelinase
treatment was without effect. Therefore, it appears that ceramide
mediates in part the inhibitory effect of GlcCer synthase inhibition on
IGF-1-induced cell proliferation in 3T3 cells. The rapid production of
decreased cyclin-dependent kinase activities by PDMP suggests that one
of the crucial sites of action of the inhibitor lies in this area.
Sphingolipids have been identified as potentially important metabolites in the regulation of cell proliferation and differentiation. Sphingoid bases, which are the precursors and breakdown products of complex sphingolipids, can inhibit protein kinase C(1) , activate cellular kinases(2, 3) , and stimulate DNA synthesis and cell division in quiescent cultures of Swiss 3T3 cells(4) . Ceramides may be liberated as a result of agonist-stimulated sphingomyelin hydrolysis and are associated with activation of cellular kinases, phosphatases, and nuclear transcription events(5) . Ceramide or a related metabolite may mediate apoptosis(6) . Glycosphingolipids have been implicated in a variety of growth-related phenomena. They may regulate receptor tyrosine kinase activity(7) , phospholipases(8) , other kinases(9, 10) , and cell-cell and cell-matrix interactions(11) .
In recent years, attempts to understand
specific cell functions of sphingolipids have been aided by the
discovery of inhibitors of sphingolipid metabolism. These include
inhibitors of 3-ketodihydrosphingosine synthase(12) , sphingoid
base acyltransferase (13) , and -glucosidase(14) .
The GlcCer (
)synthase inhibitor, PDMP, has been particularly
useful(15) . Treatment of cultured cells with the D-threo enantiomer results in the time-dependent depletion of
all the glucosphingolipids. Secondary metabolic effects include the
accumulation of ceramide and sphingosine. Inhibition of cell
proliferation is observed in concert with these changes in sphingolipid
levels(16) . In vivo studies demonstrate that PDMP
treatment inhibits tumor growth and metastasis as well as renal
hypertrophy associated with diabetes mellitus(17) .
The present study was initiated to investigate the possible role of sphingolipids in growth factor-stimulated cell proliferation. NIH 3T3 fibroblasts overexpressing receptors for insulin-like growth factor-1 (IGF-1) (18, 19) were used to examine PDMP effects on the growth factor signaling. We observed that treatment of 3T3 cells with the GlcCer synthase inhibitor abolished the proliferative response to either IGF-1 or serum. We also observed that PDMP did not affect early signal transduction events in response to serum or IGF-1, but it did block cell cycle progression in association with an inhibition of cell cycle-dependent kinases.
Ceramide was measured by spotting lipid extracts and ceramide standards (0.2-2.0 µg) on thin layer chromatography plates and separating with chloroform/acetic acid (9:1). The ceramide spots were visualized by charring(25) , and the density of the spots was quantitated using a CCD video camera, connected to a Macintosh II computer utilizing NIH Image 1.49 software.
For GlcCer measurements lipid extracts were subjected to alkaline
methanolysis by incubating each sample in 1 ml of chloroform and 0.5 ml
of 0.21 N NaOH in methanol for 1 h at 37 °C. The solution
was neutralized with 0.4 ml of 0.3 N acetic acid, vortexed,
and centrifuged at 800 g for 5 min. The lower layer
was transferred to another glass tube and dried under nitrogen. The
samples and GlcCer standards, dissolved in chloroform/methanol (2:1),
were separated with borate-impregnated high performance thin layer
chromatography plates developed with chloroform/methanol/water
(63:24:4). The lipid spots were quantitated as above.
Figure 1:
Effect of PDMP on serum and IGF-1
stimulation of thymidine incorporation. NIH-3T3 cells grown to
confluence were rendered quiescent by incubation in medium containing
0.5% serum. Cells were then incubated with no additions, with 10% serum
or with 10 nM IGF-1, in the presence or absence of 20
µM PDMP for 18 h. [H]Thymidine (1
µCi/ml) was added for 1 h, the cells were washed, and the
incorporated thymidine was measured as described under
``Experimental Procedures.'' The results are the mean
± S.E. of quadruplicate samples of a representative
experiment.
The time dependence of the inhibition of cell proliferation by the GlcCer synthase inhibitor was assessed by delaying exposure of the cells to inhibitor following the addition of IGF-1. PDMP inhibited IGF-1-stimulated thymidine incorporation, and the extent of inhibition was attenuated as the duration of exposure to PDMP was shortened (Fig. 2). These data are consistent with the interpretation that the anti-proliferative effect of PDMP is the result of secondary metabolic effects and not due to the inhibitor itself.
Figure 2:
Effect of delaying the time of PDMP
addition following IGF-1 treatment on thymidine incorporation.
Confluent cells grown to quiescence in serum-deprived medium were
incubated for a total of 20 h with 10 nM IGF-1 with or without
PDMP. PDMP (final concentration 20 µM) was added along
with IGF-1 or at different times after IGF-1 addition.
[H]Thymidine incorporation was determined as
described under ``Experimental Procedures.'' Results,
expressed as percent of IGF-1, are the mean ± S.E. of
quadruplicate samples of a representative
experiment.
Figure 3:
IGF-1 receptor phosphorylation in cells
treated with or without PDMP. Confluent cells grown to quiescence in
serum-deficient medium were treated for 18 h with or without 50
µM PDMP. The cells were then incubated with 10 nM IGF-1 for 5 or 30 min, following which they were lysed and equal
amounts of lysate protein were loaded on 7.5% SDS-polyacrylamide gels.
The proteins containing phosphotyrosine were detected by Western
immunoblotting as described under ``Experimental
Procedures.'' The results are representative of four experiments. Lane 1, control untreated cells; lane 2, control
cells incubated with IGF-1 for 5 min; lane 3, PDMP-treated
cells, incubated with IGF-1 for 5 min; lane 4, control cells
incubated with IGF-1 for 30 min; lane 5, PDMP-treated cells
incubated with IGF-1 for 30 min. Arrow points to the 97-kDa
phosphotyrosine-containing protein representing the IGF-1 receptor
subunit.
Similarly, 24 h of pretreatment with 50 µM PDMP had no discernible effect on IGF-1 stimulation of MAP kinase (Fig. 4A) and PI-3-kinase (Fig. 4B) activities. These results clearly indicated that these initial events in growth factor action were not altered by PDMP pretreatment.
Figure 4: Effect of PDMP on IGF-1 stimulation of MAP kinase activity (A) and PI-3-kinase activity (B). Confluent cells grown to quiescence in low serum medium were treated with or without PDMP for 18 h. Cells were then incubated with or without IGF-1 for 5 min and lysed with detergent-containing lysis buffers, and the enzyme activities were determined as described under ``Experimental Procedures.'' The results, representative of two experiments each, are the mean ± S.E. of quadruplicate samples in each group.
Figure 5:
Flow cytometric analysis of cell cycle
distribution in asynchronous cells treated with PDMP. Cells were plated
at an initial density of 5 10
/100-mm dishes in
medium containing 10% fetal bovine serum for 24 h. The medium contained
0 or 50 µM PDMP for 24 h as shown. A, control
cells; B, cells treated with PDMP for 24 h just before
addition of BrdU; and C, cells treated with PDMP for 24 h were
incubated for an additional 24 h in control medium. Cells from all
treatments were fixed at the same time. The dot-plots show the
simultaneous analysis of DNA synthesis by BrdU labeling to delineate
the S phase and DNA content by propidium iodide labeling. Three
discreet populations of cells representing G
/G
(2 N DNA with no BrdU incorporation (1),
S-phase (variable DNA content showing BrdU incorporation) (2),
and G
/M (4 N DNA content with no BrdU labeling) (3) are seen. The quantitation of cells in the three areas is
given in the adjoining table.
The time dependence of the PDMP effect on
cell cycle arrest in asynchronously growing cells was studied. A
time-dependent increase in the percentage of cells in G/M
with a concomitant decrease in cells in the S phase was observed (Fig. 6A). These changes occurred in a parallel manner
consistent with a block at both G
/S and G
/M.
PDMP treatment for as little as 2 h resulted in significant changes in
the distribution of cells. The peak effect was observed by 12 h with
the percentage of cells in the G
/M phases increasing by
>40% and the percentage of cells in the S phase decreasing by
>50%.
Figure 6:
Time course of PDMP effects on an
asynchronous population of cells. The cells were treated without or
with 50 µM PDMP for the indicated periods. All treatment
groups were incubated simultaneously with BrdU and processed for flow
cytometry. The results are the mean ± S.E. from three or four
experiments, except for the groups of control and 24-h PDMP-treated
cells, which had 11 experiments each. Results are expressed as percent
of untreated control cells in each of the cell cycle stages. All the
values are significantly different from control at p < 0.05
by unpaired t test. A, percent of cells in each cell
cycle stage. B, relative concentrations of ceramide and GlcCer
in cells treated as in the above experiment. Ceramide and GlcCer levels
in control cells were 1.6 ± 0.15 and 1.52 ± 0.04
µg/mg protein, respectively. Results, expressed as percent of
control, are the means from two experiments each. The fraction of cells
in G/G
, G
/M, and S phases under
control conditions were 85, 8.2, and 6.6%, respectively. This did not
vary significantly in cells control cells at 48
h.
Cellular levels of ceramide and GlcCer were measured in parallel cultures treated similarly. A representative thin layer chromatography plate showing the separation of these lipids is shown in Fig. 7. The presence of N-acetylsphingosine can be seen in cells treated with this C2-ceramide in both solvent systems, demonstrating its cellular incorporation. Time-dependent increases in cell ceramide were observed, with levels increasing more than 3-fold by 24 h (Fig. 6B).
Figure 7: Thin layer chromatograms of lipids from NIH-3T3 WT21 cells. Panel A shows separation of ceramides from other lipids with chloroform/acetic acid (9:1). Panel B shows the separation of GlcCer with chloroform/methanol/water (63:24:4). Lanes A-F, ceramide and GlcCer standards, 0.2, 0.5, 0.75, 1.0, 1.5, and 2.0 µg. Lipid extracts from cells grown in low serum for 24 h and treated for 18 h as follows: lane 1, control; lane 2, IGF-1; lane 3, IGF-1 + PDMP; lane 4, IGF-1 for 18 h + PDMP for the first 6 h only; lane 5, IGF-1 + sphingomyelinase; lane 6, IGF-1 + acetylsphingosine. Arrows point to N-acetylsphingosine (N-Acsph). Lipid standards were comprised of type III ceramide (Sigma) and GlcCer obtained from Gaucher spleen.
GlcCer levels decreased by greater than 70% at 24 h. These changes in sphingolipid levels were consistent with the well-characterized ability of PDMP to inhibit GlcCer synthesis(15) . Cell protein/dish over the same time period decreased as well (not shown).
The possible association between
cell cycle block and increased ceramide levels was examined further in
synchronized cells (Fig. 8, A and B). Cells
were rendered quiescent by serum deprivation and then stimulated with
10 nM IGF-1. Treatment with IGF-1 for 18 h resulted in an
almost 4-fold increase of cells in S phase, with a concomitant
reduction in G/G
phase, and a small, but
significant increase in G
/M cells. Addition of PDMP with
IGF-1 blocked the increase in S phase cells, the majority of cells
being arrested in G
/G
. Neither N-acetylsphingosine nor sphingomyelinase treatment inhibited
entry of cells into the S phase. In contrast to the asynchronously
growing cells, the G
/M population displayed no significant
difference in the presence or absence of PDMP or N-acetylsphingosine.
Figure 8: Cell cycle distribution (A) and cellular sphingolipid levels (B) in serum-deprived cells treated with IGF-1 ± PDMP, C2-ceramide, or sphingomyelinase. Cells rendered quiescent by serum deprivation for 24 h were treated 18 h longer with 10 nM IGF-1 in the presence or absence of 25 µM PDMP, 10 µM acetylsphingosine, or 4 milliunits/ml sphingomyelinase (SMase). The results of the cell cycle study (mean ± S.E. of three to six experiments) are presented as percent of cells in the indicated cell cycle stages in each treatment group. The ceramide and GlcCer levels (mean ± S.D. of two experiments) are expressed as percent of untreated control, the control values being 2.17 ± 0.64 and 1.72 ± 0.5 µg/mg protein, respectively.
The effect of newly described
inhibitors of GlcCer synthase on the cell cycle distribution was also
determined. BML-130 is the DL-analog of threo-PDMP in which
pyrrolidine and palmitic acid substitute for the morphiline and
decanoic acid groups, respectively(31) . IV-231B is the D-enantiomer of an analog of BML-130 in which the benzene ring
has been replaced by the alkenyl chain of sphingosine(32) . The
latter compound inhibits GlcCer synthase, depleting cells of GlcCer,
but does not result in ceramide accumulation(31) . Treatment
with each inhibitor resulted in the accumulation of cells in
G/M; however, only PDMP treatment resulted in a decrease in
the number of cells in the S phase (Table 2).
Figure 9:
Flow cytometric dot plots of asynchronous
population of cells treated for 16 h with 4 µM aphidicolin (panel B), 10 mM hydroxyurea (panel C), or
2.5 µM nocodazole (panel D). Panel A shows control cells. Percentage of cells in
G/G
, S and G
/M (areas marked 1-3, respectively) are shown in the adjoining
table.
The cell
cycle distribution was studied when cells were released from this
blockade in the presence or absence of PDMP (Fig. 10). In each
case release from blockade by control medium resulted in recovery of
the S phase. Following blockade with aphidicolin and hydroxyurea, PDMP
treatment resulted in an increase in cells at G/G
and G
/M and a decrease in cells at the S phase. In
contrast, PDMP treatment following release from mitotic arrest by
nocodazole resulted in a block at G
/S with no change at
G
/M.
Figure 10:
Effect of PDMP on cell cycle distribution
of cells synchronized by pretreatment with aphidicolin (APH, panel
B), hydroxyurea (HU, panel C), or nocodazole (NOCO,
panel D). Asynchronous population of cells were treated with or
without (C, panel A) the above agents as mentioned in
the legend for Fig. 10. After 16 h, the additions were removed
by washing three times with medium, and the cells were continued to
incubate for an additional 24 h in (1) control medium or (2) with 50 µM PDMP. The percentages of cells
(mean ± S.D.) in G/G
, S and
G
/M (areas marked 1-3, respectively) are
shown in the adjoining table. The flow cytometric dot-plots are
representative of two or three experiments.
Figure 11:
p34 kinase
activities and immunoblots. A, time course of
p34
kinase activity following treatment of
asynchronous cells with PDMP (50 µM) or sphingomyelinase
(4 milliunits/ml). Cdc2 kinase activity was measured in crude cell
lysates using the peptide substrate as described under
``Experimental Procedures.'' Results expressed as percent of
control are the mean ± S.E. of three to five experiments. The
specific activity of cdc2 kinase in control lysate was 275 ± 17
pmol [
P]/mg protein. *p < 0.05 versus control. B, immunoblot of
p34
kinase in cell lysates from the above
experiment. The cell lysates were from the following treatments: lane 1, control; lanes 2 and 5, PDMP 20 h; lane 3, PDMP 1 h; lane 4, PDMP 2 h; and lane
6, sphingomyelinase (SMase), 20
h.
The activity of cdk2 and cdc2 kinases was determined by histone
phosphorylation following immunoprecipitation with specific antibodies.
PDMP treatment for 2 or 20 h of asynchronous cells caused a reduction
in the activities of both cdc2 and cdk2 kinases (Fig. 12A). Similarly, treatment with hydroxyurea
(which arrests cells in G/S) also reduced the activity of
both these enzymes. In contrast, treatment with nocodazole (which
arrests cells in metaphase) resulted in an increase in cdc2 activity
and a decrease in cdk2 activity (Fig. 12A).
Figure 12:
Activities of cyclin-dependent kinases. A, asynchronous cells grown in serum-containing medium were
treated for 2 or 20 h with 50 µM PDMP, or for 20 h with
hydroxyurea (HU, 1 mM), or nocodazole (Noco,
2 µM). B, cells grown in serum-deprived medium
for 24 h were treated with 10 nM IGF-1 in the presence or
absence of 50 µM PDMP or 4 milliunits/ml sphingomyelinase
for 18 h. Following the treatments, cells were lysed and the lysates
subjected to immunoprecipitation using specific antibodies to
p34 kinase (cdc2) and cdk2 kinase. The kinase
activity of the immune complex was determined by histone H1
phosphorylation as described in the text. The results (percent of
corresponding untreated control) represent the mean ± S.E. of
three experiments, each sample assayed in duplicates.The specific
activity of cdk2 kinase in control cells grown in medium with and
without serum were 12.5 ± 1.3 and 5.8 ± 1.6 pmol
[
P]/mg protein, respectively. Similarly, the
specific activity of cdc2 kinase in control cells grown in medium with
and without serum were 2.8 ± 0.16 and 1.6 ± 0.3 pmol
P/mg protein, respectively. *p < 0.05 versus corresponding control by unpaired t test. C, immunoblots of cdc2 kinase. Western blotting analysis with
cdc2 antibody was done as described in the text. The lysates were from
the same treatments as described in B: lane 1,
control; lane 2, IGF-1; lane 3, IGF-1 + PDMP;
and lane 4,: IGF-1 +
sphingomyelinase.
The effect of PDMP on the cell cycle-dependent kinases was also studied in serum-deprived cells treated with IGF-1. The activity of both cell cycle-dependent kinases was increased almost 2-fold over control by IGF-1 treatment (Fig. 12B). Addition of PDMP along with IGF-1 significantly decreased these cell cycle-dependent kinase activities, while treatment with sphingomyelinase had no effect on the IGF-1-induced increase, consistent with the cell cycle data. Analysis of cdc2 by Western blotting (Fig. 12C) indicated that IGF-1 treatment for 18 h caused an induction of cdc2 kinase as shown by an increase in the 34 kDa band (lane 2, 2.6 ± 0.5-fold over control, lane 1). Inclusion of PDMP along with IGF-1 totally abolished this increase in cdc2 kinase (lane 3versuslane 2); sphingomyelinase addition along with IGF-1 had no effect on cdc2 (lane 4versuslane 2). Treatment of cells with 10 µMN-acetylsphingosine decreased the cdc2 kinase and cdk2 kinase activities to 76 and 88% of IGF-1-stimulated control values, respectively (data not shown). Similar analysis of cdk2 by Western blotting showed no change following the above treatments (data not shown).
Figure 13: Northern blot analysis of cdc2 mRNA expression. Asynchronous population of NIH-3T3 cells were incubated without (control) or with 50 µM PDMP for periods as indicated below. The total RNA was isolated and examined by Northern analysis as described under ``Experimental Procedures'' with cdc2 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fragments. Lane 1, PDMP, 1 h; lane 2, control, 1 h; lane 3, PDMP, 6 h; lane 4, control, 6 h; lane 5, PDMP, 12 h; lane 6, control, 12 h; lane 7, PDMP, 24 h; lane 8, control, 24 h.
Primary events following binding of various polypeptide growth factors, including IGF-1, to a family of receptors are the activation of protein kinases associated directly or indirectly with these receptors(37) . On binding, the kinases are activated to trigger a cascade of phosphorylation processes, culminating in DNA synthesis and cell proliferation.
In the present study we have
investigated whether alterations in endogenous sphingolipids affect
growth factor signaling via IGF-1. GlcCer synthase inhibition by PDMP
blocked serum- or IGF-1-stimulated proliferative response as indicated
by a decrease in [H]thymidine incorporation in
3T3 cells overexpressing the IGF-1 receptors. However, early
IGF-1-stimulated signaling events, including receptor
autophosphorylation, activation of MAP/basic protein kinase, and
PI-3-kinase were unaffected. Thus, the antiproliferative site of action
of PDMP must lie downstream to these early phosphorylation events.
The cell cycle analyses demonstrated that PDMP induced a specific
cell cycle block at G/S in both asynchronously growing
cells and in cells synchronized in G
by serum deprivation.
Release from cell cycle arrest with nocodazole also resulted in
G
/S blockade. PDMP treatment also caused an additional
block at G
/M.
The PDMP-induced inhibition of cell cycle
progression was reversible; cells released from the block were able to
re-enter the cycle, as shown by the recovery of S phase cells. The
antiproliferative effects of PDMP on IGF-1-stimulated proliferation
were time dependent; [H]thymidine incorporation
was reduced even when the GlcCer synthase inhibitor was added several
hours after IGF-1. However, PDMP was most effective in blocking the
proliferative response when added simultaneously with the growth factor
and became less effective when its addition was delayed. The effect of
PDMP was therefore not due to direct inhibition of
[
H]thymidine uptake, but probably due to
time-dependent changes in sphingolipid levels.
The growth inhibitory effects of PDMP were accompanied by a time-dependent increase in cell ceramide and a decrease in GlcCer levels. This is consistent with our earlier interpretation that blockade of GlcCer synthase is the cause of growth inhibition(15) . Cellular ceramide levels were also raised by sphingomyelinase; interestingly, C2-ceramide treatment also increased the endogenous ceramides, a finding previously observed in MDCK cells treated with a longer chain ceramide, octanoylsphingosine(38) . C2-ceramides are readily incorporated into cultured cells, and they have therefore been used extensively as a test of the biological activity of ceramide(39) . However, the secondary effects of short chain ceramides on the metabolism of endogenous sphingolipids, including glycosphingolipids and sphingomyelins, require that the use of these compounds be interpreted cautiously. A correlation was observed between PDMP effects on cell cycle changes and ceramide accumulation. Cell cycle inhibition was not seen when cell ceramide levels were increased by incubation with sphingomyelinase but was seen with C2-ceramide treatment.
Recently,
a second messenger function for ceramide has been proposed. In this
model the activation of a neutral sphingomyelinase by agonists
generates ceramide(40) , which mediates the functional response
to these agents (see (5) ). For example, in the leukemia cell
line HL-60, ceramide has been suggested to mediate many of the effects
of tumor necrosis factor-, such as cell growth and
differentiation(5) , programed cell death (or
apoptosis)(6) , and activation of the nuclear factor
B(41) . However, other recent studies have shown that not
all effects of tumor necrosis factor-
are mediated by
ceramide(42, 43) . A recent study (44) has
reported that treatment of T lymphocytes with agents such as
interleukin-2 or phorbol ester plus ionomycin decreased the ceramide
levels and increased [
H]thymidine incorporation,
while PDMP, which increased endogenous ceramide, inhibited
[
H]thymidine incorporation observed at 24 h or
more. Furthermore, C2-ceramide and sphingosine analogs had less
pronounced growth inhibitory effects, since a decrease in
[
H]thymidine incorporation could be seen only at
48, not in 24 h. Consistent with our observations, sphingomyelinase
treatment, which also increased ceramide levels, did not inhibit T-cell
growth. Based on these data, it was hypothesized that there may be
functionally distinct pools of ceramide within the T cell.
Several
studies have reported that the addition of some sphingolipids, such as
ganglioside GM, results in inhibition of cell
proliferation; conversely, metabolites of gangliosides may enhance
proliferation. The effects of gangliosides in these studies have been
correlated with the regulation of receptor kinase activities associated
with the platelet-derived growth factor, epidermal growth
factor(9, 10) , and insulin receptors(7) .
PDMP treatment causes depletion of cell gangliosides(15) , at a
speed depending on the rates of ganglioside turnover, and therefore
could inhibit growth factor-stimulated cell proliferation by inhibition
of receptor tyrosine kinase activity. PDMP has been reported to enhance
tyrosine phosphorylation of T cells stimulated with
interleukin-2(45) . Alternatively, ceramide has been identified
as a potential mediator of EGF receptor phosphorylation at the
threonine 669 site and therefore may regulate receptor kinase activity.
However, the absence of observable changes in IGF-1 receptor
phosphorylation, MAP kinase, or PI-3-kinase activities, suggests that
ganglioside or ceramide-mediated effects on early signaling events
cannot explain the antiproliferative effects of PDMP in 3T3 cells. Our
preliminary results also suggest that sphingosine may not be the
mediator of PDMP inhibition of cell proliferation, since addition of
sphingosine (1-5 µM) by itself was growth
stimulatory as indicated by an increase in
[
H]thymidine or BrdU incorporation. (
)
Major progress has been made in recent years in our
understanding of the regulation of cell cycle. Progression through cell
cycle transition points in eukaryotic cells is controlled by the
activation of cyclin-dependent kinases. The timing of activation of
these kinases is regulated by association with regulatory subunits
(cyclins), and by phosphorylation(34) . There now are
well-documented examples of both positive and negative control
mechanisms for these processes. In yeast, the control of
G/S and G
/M transitions is mediated by a single
cell cycle-dependent kinase in association with different cyclins,
while in mammalian cells, it is controlled by multiple cell
cycle-dependent kinases in association with multiple cyclins; each of
these complexes act at distinct cell cycle stages (for reviews, see
Refs. 46, 47)
The effect of PDMP on two kinases, the p34 kinase associated with progression of cells into mitotic phase,
and the cdk2 kinase which is activated at G
/S, was
evaluated. The activity of p34
kinase was increased when
the cells were arrested in mitosis by nocodazole treatment and markedly
reduced when the cells were arrested at G
/S by hydroxyurea.
Similar changes in cdc2 kinase activity were reported in Hela cells
following treatment with nocodazole and hydroxyurea(48) . Both
nocodazole and hydroxyurea treatments, however, reduced the cdk2
activity, probably reflecting a lack of cells in G
/S
transition.
PDMP treatment of asynchronously growing cells caused a
decrease in both the cdc2 and cdk2 kinase activities, consistent with a
cell cycle blockade occurring at G/M and G
/S
transitions, respectively. Increasing ceramide levels with N-acetylsphingosine or other PDMP analogs partly replicated
the G
/M block but not the G
/S block seen with
PDMP. Since these inhibitors all block GlcCer formation to a comparable
extent, the G
/M block is most likely due to ceramide or a
ceramide catabolite and is unlikely to be due to cellular depletion of
glycosphingolipids. Ceramide has recently been identified as a
potential activator of protein phosphatase 2a. This phosphatase
inhibits the transition from G
to mitosis by
dephosphorylating cdc25 and p34
(49) . It is
tempting to speculate that the inhibitory effects of ceramide on
G
/M transition are mediated by activation of this
phosphatase.
In conclusion, the present study demonstrates that the GlcCer synthase inhibitor PDMP has specific and reversible effects on growth factor-stimulated cell growth in NIH-3T3 cells. PDMP treatment causes cell cycle arrest, in association with an inhibition of the cell cycle-dependent kinases. Early signaling events are not affected. Although the present study does not resolve the question of the mechanism for PDMP-induced growth inhibition, our results indicate that ceramide may contribute to this effect.