(Received for publication, September 1, 1994; and in revised form, November 21, 1994)
From the
The dependence of some cell types on serum factors for growth
may represent a powerful, but poorly studied, model for antimitogenic
pathways. In this study, we examine ceramide as a candidate
intracellular mediator of serum factor dependence. In Molt-4 leukemia
cells, serum withdrawal caused a significant arrest in cell cycle
progression (80% of cells in G/G
), accompanied
by a modest apoptotic cell death (12%). Serum deprivation of these
cells resulted in significant sphingomyelin hydrolysis (72%;
corresponding to hydrolysis of 47 pmol/nmol phosphate), which was
accompanied by a profound and progressive elevation (up to
10-15-fold) in endogenous levels of ceramide. Withdrawal of serum
caused the activation of a distinct, particulate, and
magnesium-dependent sphingomyelinase. The addition of exogenous
C
-ceramide induced a dramatic arrest in the
G
/G
phase of the cell cycle comparable to the
effects observed with serum withdrawal, albeit occurring much sooner.
Unlike serum withdrawal, however, the addition of
C
-ceramide resulted in more pronounced apoptosis. Because
of the previously noted ability of exogenously added phorbol esters to
inhibit ceramide-mediated apoptosis, we investigated the hypothesis
that endogenous activation of the diacylglycerol/protein kinase C
pathway may modulate the response to serum withdrawal. Indeed, serum
withdrawal resulted in 3-4-fold elevation in endogenous
diacylglycerol levels. The addition of exogenous diacylglycerols
resulted in selective attenuation of ceramide's effects on
apoptosis but not on cell cycle arrest. Thus, the combination of
ceramide and diacylglycerol recapitulated the complex effects of serum
withdrawal on cell cycle arrest and apoptosis. These studies identify a
novel role for ceramide in cell cycle regulation, and they may provide
the first evidence for an intracellular signal transduction pathway in
mammalian cells mediating cell cycle arrest. These studies also
underscore the importance of lipid second messengers and the
significance of the interplay between glycerolipid-derived and
sphingolipid-derived lipid mediators.
Serum deprivation is a powerful mechanism by which cells can be
arrested in the cell cycle and induced to undergo programmed cell death (1, 2, 3) . As such, the serum deprivation
model has been used extensively to study both of these events. However,
a mechanistic basis for this growth arrest and apoptosis has yet to be
found. With the discovery of the sphingomyelin cycle,
sphingolipid-derived molecules have gained prominence as critical
antimitogenic molecules of cells(4, 5) . Ceramide, a
product of regulated sphingomyelin hydrolysis, has been shown to be
released within minutes to hours following stimulation of cells with
agents such as 1,25-dihydroxyvitamin
D
(6, 7) , tumor necrosis factor
(8, 9) ,
interleukin-1
(10, 11) , and
-interferon(8) . Agonist-induced mobilization of ceramide
has been found to precede the antimitogenic effects of the agonists,
and exogenous addition of short chain membrane-permeable ceramides has
been shown to reproduce the antiproliferative and differentiative
effects of these agonists(7, 12) . Furthermore, in the
case of tumor necrosis factor
, ceramide has been implicated as a
mediator of programmed cell death(13) . These studies suggested
that the antiproliferative effects of ceramide may be a consequence of
apoptosis; however, an effect of ceramide on cell cycle progression has
not been examined. Prompted by these findings and considerations, we
investigated a role for ceramide in mediating the growth suppressive
effects of serum deprivation.
In this study, we show that ceramide
levels respond progressively to serum withdrawal through activation of
a magnesium-dependent, membrane-associated, neutral sphingomyelinase.
Ceramide is found to induce a significant block in cell cycle
progression accompanied by apoptosis. Interestingly, diacylglycerol
(DAG)(), which also increases with serum withdrawal,
counters the effects of ceramide on apoptosis but not on cell cycle,
suggesting a protective role for DAG. More importantly, the combination
of ceramide and DAG recapitulates the effects of serum withdrawal on
cell cycle arrest. The implications of these studies on the role of
ceramide in cell cycle arrest are discussed.
H NMR
spectra were obtained on a G.E. 500-MH
Omega spectrometer.
Chemical shifts (
) were indicated as parts/million relative to a
TMS internal standard. Mass spectra were obtained using a
Hewlett-Packard 5988 GO/MS/DS system. The samples were analyzed using
chemical ionization mass spectrometry. MS is m/z, 398
(M
);
H NMR (CD
OD)
is as
follows: 0.84 (6H, dt, CH
-C(17), CH
-C(6`)),
1.25 (26H, sbr, CH
), 1.38-1.42 (2H, m,
CH
), 1.52-1.62 (2H, m, CH
),
2.00-2.08 (2H, m, CH
-C(6)), 2.14-2.22 (2H, t,
CH
-C(2`)), 3.67 (2H, d, H
C(1)), 3.83-3.87
(1H, m, HC(2)), 4.04 (1H, t, HC(3)), 5.42-5.50 (1H, m, HC(4),
5.62-5.72 (1H, m, HC(5)), 7.66-7.72 (1H, m, NH).
DL-Erythrodihydro-C-ceramide was prepared via a
similar procedure using DL-erythrodihydrosphingosine (Sigma
D7033).
Molt-4 T leukemia cells were found to require serum factors
for growth in tissue culture; thus, withdrawal of serum factors led to
both an arrest of cells in the G/G
phase of the
cell cycle and programmed cell death (PCD) (Fig. 1). Cell cycle
arrest was observed as early as 12 h following serum withdrawal. By 26
h, cell cycle arrest was pronounced, with almost 80% of cells in the
G
/G
stage as opposed to 61% in control
cultures. By 48 h of serum starvation, very few cells could be found in
either the S (2%) or the G
/M (2%) phases, and nearly all
cells had arrested in G
/G
. Some cells also
underwent PCD, which was observed by 26 h. By 48 h following serum
deprivation, a significant proportion (12%) of cells appeared in a
pre-G
/G
, apoptotic peak (Fig. 1, A and C). Therefore the growth arrest observed with serum
deprivation involves predominantly cell cycle arrest with a small
component of apoptosis.
Figure 1: Effects of serum deprivation on cell cycle and PCD. Molt-4 cells were grown with or without 10% fetal calf serum (FCS) for the indicated periods of time. Cells were fixed, stained, and analyzed as described under ``Methods.'' A, representative tracings from FACS analyses; B and C, quantitative measure of FACS data using the sum of broadened rectangles (SOBR) model.
Since little insight is available concerning
the mechanisms for the antimitogenic effects of serum factor
withdrawal, we initially wondered whether ceramide, which has been
implicated as an intracellular mediator of tumor necrosis factor
-induced apoptosis(13) , may play a role. Therefore, the
response of endogenous ceramide to serum withdrawal was investigated.
Serum starvation of Molt-4 cells led to a remarkable increase in
endogenous ceramide (Fig. 2A). A 3-fold increase in
ceramide levels could be observed as early as 24 h following the
removal of serum. This initial elevation was comparable to
agonist-induced ceramide elevations described in other cell systems.
However, unlike other agonist-stimulated systems where ceramide levels
returned to basal values(6, 7) , prolonged serum
starvation produced further elevations in ceramide. Thus, by 48 h an
8-fold increase in ceramide could be observed, and by 96 h ceramide had
increased further to 15-fold above basal. Since ceramide levels were
normalized for total phospholipid levels, these results reflected
specific increases in ceramide levels. These increases in ceramide
coincided with both the cell cycle arrest and PCD elicited by serum
deprivation.
Figure 2:
Effects of serum deprivation on
sphingomyelin metabolism. Molt-4 cells were serum-starved for the
indicated time periods and analyzed as described under
``Methods.'' The results shown are representative of two to
five separate experiments. A, time course of ceramide
elevations; B, mass of sphingomyelin following 72 h of
treatment; C, sphingomyelinase activity from cells treated for
72 h. Neutral sphingomyelinase activity in the presence of
Mg is shown. No activity was observed in the absence
of Mg
.
Since the kinetics and magnitude of the ceramide
response were sufficiently distinct and more pronounced than those
previously observed with tumor necrosis factor and other
extracellular cytokines, it became important to determine the source of
ceramide as well as the mechanisms involved in generating ceramide. The
elevation in ceramide levels was paralleled by a concomitant decrease
(from 64 to 17 pmol/nmol phosphate) in the mass of sphingomyelin (Fig. 2B). This loss in sphingomyelin (47 pmol/nmol
phosphate at 72 h) accounts for most of the generated ceramide at the
same time point (38 pmol/nmol phosphate). However, investigation of the
mechanism of hydrolysis of sphingomyelin revealed that serum withdrawal
did not induce any substantial activity in the
cytosolic/magnesium-independent, neutral sphingomyelinase (Fig. 2C), which has been implicated previously in the
mechanism of action of 1
,25-dihydroxyvitamin
D
(22) . On the other hand, withdrawal of serum
factors resulted in substantial activation of a particulate,
magnesium-dependent, neutral sphingomyelinase with an increase from
10,000 to 24,000 cpm/mg protein (Fig. 2C). Thus, serum
withdrawal appears to stimulate a signaling cascade distinct from
agonist stimulation of the sphingomyelin
cycle(6, 7, 8, 9, 10, 11) .
However, the outcome of both pathways is the induction of sphingomyelin
hydrolysis and ceramide generation, although the magnitude and
persistence of the ceramide response upon serum withdrawal reaches
levels and durations not seen with agonist stimulation.
We next
investigated what role ceramide may play in mediating the effects of
serum deprivation. A short chain synthetic ceramide,
C-ceramide, was employed to treat Molt-4 cells in the
presence of serum. Addition of C
-ceramide not only caused
PCD (Fig. 3), it also induced significant cell cycle arrest.
Ceramide-induced cell cycle arrest occurred rapidly (within 4 h) and
doses as low as 15 µM C
-ceramide
(approximately 4 nmol/10
cells; comparable to doses
achieved upon serum deprivation) were able to reproduce effects
reminiscent of prolonged serum deprivation: almost complete
G
/G
arrest following 14 h of treatment. Higher
doses (20-40 µM) were more rapidly effective (within
4 h) and lower doses (10 µM) exhibited decreased efficacy,
illustrating that ceramide's effects on both PCD and cell cycle
exhibited time and dose dependence (Fig. 3, B and C). Furthermore the effects of ceramide on cell cycle arrest
was found to be a more generalized phenomenon, not restricted to the
transformed Molt-4 cell line. In a number of non-transformed cells,
including the human Wi38 human diploid fibroblast line, 15 µM C
-ceramide was able to produce substantial cell cycle
arrest in a time-dependent fashion (Fig. 3D).
Figure 3:
Effects of C-ceramide and
dihydro-C
-ceramide on cell cycle and PCD. Molt-4 cells and
Wi38 cells were grown as indicated in ``Methods.'' Cells were
treated with C
-ceramide, dihydro-C
-ceramide, or
ethanol vehicle for the indicated times. Cells were prepared and
analyzed as described under ``Methods.'' A,
representative FACS tracings at 14 h; B-F, quantitative
measure of FACS data using the SOBR model (B and C,
Molt-4 cells treated with C
-ceramide; D, Wi38
cells treated with 15 µM C
-ceramide; E and F, dihydro-C
-ceramide
treatments).
Next,
it became important to establish the specificity of ceramide action.
Thus, Molt-4 cells were treated with the closely related lipid
molecule, dihydroceramide, a biosynthetic precursor for ceramide.
Dihydroceramide differs structurally from ceramide in only one respect;
it lacks the double bond between carbons 4 and 5 of the sphingoid
backbone. In Molt-4 cells, dihydro-C-ceramide treatment, at
concentrations and times comparable to those that were effective with
C
-ceramide, proved to be ineffective at producing either
cell cycle arrest or PCD (Fig. 3, E and F),
although both molecules show similar kinetics of uptake and minimal
metabolism(23) . Thus, the differences in biologic activity are
not a result of differential delivery, but probably reflect specific
interaction of ceramide with an intracellular target that does not
interact with dihydroceramide. A possible candidate is
ceramide-activated protein phosphatase, which is specifically activated in vitro by C
-ceramide and not by
dihydro-C
-ceramide(24) . Furthermore, the
specificity of action of ceramide was also supported by the lack of
effect of diacylglycerol (a structurally related molecule) on cell
cycle progression (see below).
Thus, ceramide appears to regulate specifically the biological effects of serum deprivation. However, one critical difference between the biology elicited by ceramide and that elicited upon serum starvation related to the more pronounced effects of ceramide at stimulating PCD (compare Fig. 1C and Fig. 3C). This observation raised the possibility that other factors may operate in modulating cell viability following serum deprivation. In U937, HL60, L929/LM, and WEHI 164/13 cells, phorbol esters (activators of protein kinase C) have been noted to oppose the effects of ceramide on PCD(13, 25) . We therefore investigated whether diacylglycerols (endogenous activators of protein kinase C) may play a role in the response to serum deprivation.
During serum deprivation of Molt-4 cells, DAG levels were found to become elevated (Fig. 4A). By 24 h following serum starvation, DAG levels increased 1.5-fold (whereas ceramide levels had tripled). By 96 h, DAG levels increased by a total of 4-fold (Fig. 4A), demonstrating a progressive build-up of DAG levels, albeit to a level significantly less than that seen with ceramide (4-fold versus 15-fold at 96 h; compare Fig. 2A and Fig. 4A).
Figure 4:
Effects of serum deprivation on endogenous
diacylglycerol levels and effects of diacylglycerol on
C-ceramide-induced PCD and cell cycle arrest. A,
Molt-4 cells were serum-starved for the indicated time periods. Cells
were harvested, and the lipids were analyzed as described under
``Methods.'' The results shown are representative of five
separate experiments. B, Molt-4 cells, grown in 2% FCS, were
treated with dioctanoylglycerol plus C
-ceramide or ethanol
for the indicated times. Cells were prepared and analyzed as described
under ``Methods.''
We therefore
investigated if the simultaneous increase in both ceramide and DAG
could explain the decreased effect of serum deprivation on PCD. To test
this hypothesis, a cell-permeable diacylglycerol, dioctanoylglycerol
(diC), was employed, both alone and in combination with
ceramide for impact on cell cycle and PCD. Alone, diC
, at
concentrations of 10 to 100 µM, was ineffective at causing
either cell cycle arrest or PCD during 5-30 h (data not shown).
However, in combination with C
-ceramide diC
reversed the apoptotic effects of ceramide by more than 50% (Fig. 4B). In a quantitative analysis, diC
and phorbol esters also inhibited ceramide-induced DNA
fragmentation and loss of viability. (
)In contrast,
diC
exhibited minimal effects in reversing ceramide-induced
cell cycle arrest (at best, a 10-15% reversal was observed).
These data suggest that the increase in endogenous DAG may account for
the decreased PCD observed with serum deprivation compared to that
found upon ceramide stimulation. Importantly, ceramide and
diacylglycerol, together, faithfully recapitulated the effects of serum
withdrawal on growth and viability.
These studies identify a novel
role for ceramide in regulating cell cycle progression whereby ceramide
induces a significant G/G
arrest. This
observation carries several implications. First, ceramide may emerge as
an endogenous mediator of cell cycle arrest. As opposed to the myriad
of candidate intracellular messengers and mediators of cell cycle
progression, little is known on the operation of intracellular
mediators of cell cycle arrest. Thus, these studies identify an
important candidate for this role. This function for ceramide is likely
in the context of serum withdrawal where endogenous ceramide levels
accumulate to very high levels, and where exogenous ceramide induces
very early effects on cell cycle progression. Further examination of
this hypothesis awaits the development of specific inhibitors of
ceramide generation and/or action. Second, these studies raise the
tantalizing possibility that ceramide modulates the endogenous
machinery regulating cell cycle progression. Indeed, in ongoing
studies, support for such a role is provided by the ability of ceramide
to induce dephosphorylation of the retinoblastoma gene product (
)with specificity, potency, and kinetics that match the
ability of ceramide to induce cell cycle arrest. Third, the ability of
diacylglycerol to oppose ceramide's effects on PCD preferentially
over cell cycle arrest distinguishes these two outcomes of ceramide
action as products of distinctly-regulated mechanisms. This raises the
possibility that ceramide may function as a proximal sensor and
transducer of cell deprivation/insult/injury with the ability to launch
distinct programs of cell suppression (growth arrest and apoptosis),
the outcome of which may be dependent on whether other modulatory
signals (such as diacylglycerol) are also activated. Finally, this
latter finding suggests that sphingolipid and glycerophospholipid
signaling may be coupled or interrelated, with the opposing effects of
ceramide and DAG of greater relevance in defining the outcome on growth
and viability. A perturbation of this ratio, whether by changes in
ceramide, DAG, or both, could therefore lead to changes in cell growth
and viability.
Previous studies have identified ceramide and DAG as rapidly mobilized second messenger molecules in the sphingomyelin and phosphatidylinositol cycles, respectively(6, 7, 8, 9, 11, 26, 27, 28, 29) . With DAG, evidence is accumulating on more sustained DAG signals generated from either the phospholipase D pathway (30) or through de novo biosynthesis(31) . The current study shows that the role of ceramide (and DAG) in cell cycle arrest and PCD not only exhibits slower kinetics, it occurs over a prolonged period resulting in very high levels of accumulation. Moreover, the mechanism involved in this long term generation of ceramide (magnesium-dependent, particulate sphingomyelinase) is distinct from that operating in cytokine activated sphingomyelin cycle (magnesium-independent, cytosolic sphingomyelinase) and may represent the counterpart of phospholipase D in long term DAG signaling. This description of ceramide and DAG as long term effector molecules suggests a different perspective on lipid mediators. According to this hypothesis, sustained changes in ceramide and DAG levels (probably by distinct mechanisms, as shown here for ceramide) may induce long term, and perhaps permanent, reprogramming of cell function through the regulation of apoptotic and cell cycle machinery.