Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
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ABSTRACT |
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Two immortalized cell lines, sup (+)
and sup (), derived from mutagenized Syrian hamster embryo cells,
were used to study the relationship and temporal order between calcium
and ceramide signals during apoptosis. The early preneoplastic cells,
termed sup (+), suppress tumorigenicity when hybridized with tumor
cells, whereas later-stage sup (
) cells do not. In reduced serum
conditions, sup (+) cells cease proliferating and undergo apoptosis; in
contrast, sup (
) cells continue slow growth and undergo necrosis. In
sup (+) cells, decreased endoplasmic reticulum (ER) calcium occurs 4 h after low serum treatment and precedes apoptosis. Significant elevations in ceramide are observed 16 h after reduced serum
treatment of sup (+) cells but are not found in sup (
) cells.
Inhibiting ER calcium depletion in low serum-treated sup (+) cells by
treating with high levels of calcium prevents both ceramide generation and apoptosis. Conversely, inducing ER calcium depletion in sup (
)
cells by treating with low serum plus thapsigargin results in elevated
ceramide levels and apoptosis. Furthermore, C6-ceramide treatment induced apoptosis of sup (
) cells in low serum, a condition that does not normally cause apoptosis. C6-ceramide
treatment did not induce apoptosis in either sup (+) or sup (
) cells
in 10% serum but did cause G2/M arrest. These studies show
that ceramide production is downstream of ER calcium release.
endoplasmic reticulum; thapsigargin; cell cycle arrest; diacylglycerol
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INTRODUCTION |
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A ROLE FOR CERAMIDE
in apoptosis was first defined in U-937 cells where it was shown that
tumor necrosis factor- produced an early elevation in ceramide
before apoptosis (23). Obeid and coworkers
(23) found that exogenous ceramide treatment could induce
apoptosis independent of other apoptotic stimuli. The effect of
ceramide was found to be specific in that a closely related lipid
molecule (dihydroceramide) and other lipid second messengers [diacylglycerol (DAG) and sphingosine] were unable to induce
apoptosis. Since these initial studies, other inducers of apoptosis
have also been associated with ceramide production, including Fas
(5, 37), ultraviolet (40) and ionizing
radiation (6), heat shock (43), oxidative
stress (9), and nutrient reduction (11).
Two different kinetics of ceramide elevation have been observed in response to apoptotic stimulation of cells. Several studies have suggested that ceramide elevation occurs early in the apoptotic process, with the peak response evident within 5-30 min (10, 14, 19). Other studies, using different cell types and stimuli, have shown ceramide elevations occurring many hours after treatment, paralleling late events of apoptosis (11, 37). In fact, in another model of serum restriction-induced apoptosis, the MOLT-4 cell system, ceramide elevations were found to occur 24-96 h after treatment, concurrent with apoptosis (11).
Recent studies with a number of cell lines also implicate early changes in endoplasmic reticulum (ER) calcium with the induction of apoptosis. Baffy et al. (2) first reported that induction of apoptosis after interleukin (IL)-3 withdrawal from an IL-3-dependent cell line was associated with loss of calcium from ER stores. Other groups, including our own, have since confirmed that perturbation of ER-compartmentalized calcium is important for induction of apoptosis (3, 16, 26, 28).
To investigate mechanisms involved in apoptosis, we used two
immortalized lines derived after mutagenesis of Syrian hamster embryo
(SHE) cells, sup (+) and sup () cells. The early preneoplastic cells,
termed sup (+), suppress tumorigenicity when hybridized with tumor
cells, whereas the later-stage sup (
) cells have lost the ability to
suppress tumors. In reduced serum conditions, sup (+) cells cease
proliferating and undergo apoptosis, whereas sup (
) cells continue
slow growth and undergo necrosis. This is consistent with previous
studies that have illustrated that early neoplastic cells often show an
increased susceptibility to apoptosis that is lost in later-stage
preneoplastic cells (26). We were interested in
investigating the changes that occur during neoplastic progression that
render the cells less susceptible to apoptosis. We have previously demonstrated differences in calcium homeostasis in sup (+) vs. sup (
)
cells. When treated with low serum, the sup (+) lineage responds with
an early decrease in ER calcium followed by apoptosis; whereas, the sup
(
) lineage retains normal ER calcium levels and does not undergo
apoptosis (26). If sup (+) or sup (
) cultures are
treated with pharmacological agents that reduce ER calcium, both
lineages undergo apoptosis. Conversely, in the sup (+) lineage, if
depletion of ER calcium stores is prevented, then low serum-induced apoptosis is also prevented. These studies imply that a decrease in ER
calcium is an important step needed to transduce the low serum-induced
apoptotic response.
Recent studies have suggested an interaction between calcium signals
and ceramide signals (38, 44). Prompted by these findings,
we investigated whether ceramide elevations occur during low
serum-induced apoptosis of sup (+) cells. We report here that ceramide
elevations occur after 16-24 h of low serum treatment and follow
the early calcium changes. If we pharmacologically decrease ER calcium,
ceramide is elevated. Thus modulation of ER calcium levels leads to
downstream effects on ceramide levels. Furthermore, we find that,
although addition of C6-ceramide can induce apoptosis in
sup () cells in low serum, it is not sufficient to induce apoptosis
in 10% serum. Thus, in the absence of other stressors, ceramide
appears to signal cell cycle arrest rather than death.
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MATERIALS AND METHODS |
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Materials. RNase A was obtained from 5Prime-3Prime, and C6-ceramide and C6-dihydroceramide were purchased from Biomol Research Laboratories. All other chemicals were acquired from Sigma/Aldrich. Institute for Biological Research (IBR) medium was obtained from Life Technologies, and FCS was obtained from Intergen.
Cell lines and cell culture.
Two SHE-derived lineages, originally immortalized via asbestos
mutagenesis, were used in these studies (15, 27). The sup (+) lineage represents an early stage of tumorogenesis that has lost a
senescence gene(s) but retains tumor suppressor capability. The sup
() lineage represents a later stage of tumor progression that has
lost both senescence and tumor suppressor genes. During normal passage,
cells were maintained in Dulbecco's modified IBR medium containing
10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cultures
were maintained in a 37°C incubator with 10% CO2-90%
air. Cell lines were routinely tested and found negative for mycoplasma.
DNA fragmentation analysis. Cells were seeded in 100-mm plates at a density of 5 × 105 cells/plate and were grown for 24 h. Adherent cells were washed with calcium- and magnesium-free PBS (CMF-PBS), and 10 ml of the appropriate treatment medium were added. After 24 h, cells were harvested by scraping into treatment media and pelleted. The cell pellets were washed with CMF-PBS and pelleted and lysed in 50-100 µl of lysis buffer (10 mM EDTA, 50 mM Tris, pH 8, 0.5% sodium lauryl sarcosine, and 0.5 mg/ml proteinase K). Lysates were incubated at 50°C for 2 h, RNase was added, and the lysates were incubated at 37°C for a further 30 min. DNA was extracted and analyzed for fragmentation as described previously (12, 26).
Lipid quantitation.
Cells were seeded in 100-mm plates at a density of 5 × 105 cells/plate and were grown for 24 h. Adherent
cells were washed with CMF-PBS, and 10 ml of the appropriate treatment
media were added. After 4-24 h (see legends for Figs. 1-8),
cells were harvested by scraping into treatment media, pelleted, and
then washed with CMF-PBS. Cell pellets were resuspended in 3 ml of
chloroform-methanol (1:2), and lipids were extracted via the method of
Bligh and Dyer (4). Lipids, dried under nitrogen, were
resuspended in 100 µl of chloroform, 20 µl duplicates were used for
phosphate measurements (1), and 20-µl duplicates were
used in the DAG kinase (DGK) assay (25, 39).
Phosphorylated lipids were spotted on TLC plates, and plates were
developed in chloroform-acetone-methanol-acetic acid-water
(10:4:3:2:1). The ceramide-phosphate and phosphatidic acid spots were
scraped into scintillation fluid and counted on a Packard Minaxi
liquid scintillation counter. Ceramide-phosphate (representative of
ceramide levels) and phosphatidic acid (representative of DAG levels)
were quantitated using external ceramide and DAG standards,
accordingly. Both were normalized against measured phosphate of the
same samples.
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Cell cycle analysis.
Cells were seeded in 100-mm plates at a density of 5 × 105 cells/plate and were grown for 24 h. Adherent
cells were washed with CMF-PBS, and 10 ml of the appropriate treatment
media were added. After 16 h, cells were harvested by scraping
into treatment media and analyzed as previously described
(11). Briefly, cell pellets were completely resuspended in
1 ml of CMF-PBS, 4 ml absolute ethanol was added to fix cells, and
suspensions were stored at 20°C. On the day of analysis, the
fixation buffer was removed, the cells were resuspended in 1 ml
CMF-PBS, RNA was removed using 2 mg/ml DNase-free RNase, and cells were
stained with 10 mg/ml propidium iodide for 30 min. Cells were analyzed
using a Becton-Dickinson FACSort flow cytometer. Flow cytometric
analyses were performed using an argon laser at 488 nm, and apoptotic
cells were evident as shrunken cells containing <2 N DNA.
Statistical analyses. In comparing two groups, statistical significance was determined by Student's t-test. For multiparameter comparisons, statistical significance was determined by ANOVA, adjusting for multiple comparisons using Fisher's test for significance. A value of P < 0.05 was considered to be significant.
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RESULTS |
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Changes in ceramide are not observed after 4 h of low serum
treatment.
Previous studies using sup (+) and sup () cells have shown that low
serum treatment results in different end points in the two lineages
(27). Previous studies also have documented the time
course and mode of death of the sup (+) cells in low serum (27). In 10% serum, both sup (+) and sup (
) cells
proliferate, and cell number more than doubles within 24 h (Fig.
1). In low serum conditions, sup (+)
cells cease proliferating and undergo apoptosis (27). By
24 h, the effects of low serum on sup (+) cells are evident as a
decrease in cell number (Fig. 1). In contrast, the sup (
) line
responds to low serum by decreasing growth rate and death via necrosis.
Thus, after 24 h of low serum treatment, sup (
) cell numbers
remain similar to the baseline (time 0) numbers (Fig. 1).
Ceramide elevations occur after protracted low serum treatment.
Depending on the system, ceramide generation has been found to occur
with two different kinetics; some agents produce ceramide elevation
within seconds to minutes (14, 32), and others produce an
elevation in ceramide only after an extended time, i.e., 24-96 h
(11, 37). We therefore examined whether changes in
ceramide occur downstream of calcium changes. We find that ceramide
elevation occurs late in apoptosis in hamster cells. In contrast to the data at 4 h, 24 h of low serum treatment resulted in an
~70% elevation in ceramide in sup (+) cells compared with control,
10% serum-treated cells (Fig. 3). This
elevation in ceramide was not evident in the sup () population, which
does not undergo apoptosis after 24 h of low serum treatment.
These findings are consistent with the hypothesis that ceramide
elevation occurs exclusively in cells bound for apoptosis. Furthermore,
the late kinetics of ceramide elevation places ceramide downstream of
ER calcium changes during the apoptotic process.
ER calcium changes are upstream of ceramide elevations.
An elevation in ceramide at 24 h is consistent with a role for
ceramide downstream of decreased ER calcium in the apoptotic response.
To substantiate that ceramide is indeed part of the signaling cascade
leading from ER calcium store depletion to apoptosis, it is necessary
to show a causal relationship between depletion of ER calcium stores
and elevation of ceramide. To assess this, we used thapsigargin to
pharmacologically deplete ER calcium stores. Our previous studies
showed that thapsigargin, within 48 h, induced apoptosis of
sup (+) cells in 10% serum and sup () cells in low serum, conditions
where apoptosis does not normally occur (26, 27).
Consistent with this, we found that thapsigargin treatment for 16 h was sufficient to induce a significant ceramide elevation in both sup
(+) and sup (
) cells (Fig. 4). Sup (+)
cells treated with thapsigargin in 10% serum showed ~35% elevation
in ceramide, and sup (
) cells treated with thapsigargin in
conjunction with low serum showed ~60% elevation in ceramide.
Additionally, we showed previously that raising extracellular calcium
could prevent both the low serum-induced decrease in ER calcium and
apoptosis (26). If an increase in ceramide is downstream
of decreased ER calcium, then preventing the decrease in ER calcium
should prevent the increase in ceramide. Consistent with this
hypothesis, we found that preventing low serum-induced ER calcium
depletion in the sup (+) lineage was sufficient to prevent ceramide
elevation. Thus sup (+) cells treated with 3 mM extracellular calcium
in low serum for 16 h exhibited ceramide levels that were
comparable to control cells (Fig. 4). These data suggest that changes
in ceramide levels are downstream of decreased ER calcium levels and
that ceramide is a potential component of the signal that leads from
low serum-induced ER store depletion to apoptosis.
C6-ceramide induces apoptosis in low serum.
If ceramide is indeed an important transducer in the pathway between ER
calcium reduction and apoptosis, then not only must ceramide be
elevated after ER calcium reduction but exogenous ceramide treatment
should result in the induction of apoptosis. To determine whether
ceramide was able to induce cell death, we treated sup () cells in
0.2% serum with synthetic C6-ceramide and the inactive
ceramide analog C6-dihydroceramide for 16 h. Addition
of C6-ceramide caused a 35% decrease in cell number, whereas treatment with the inactive analog
C6-dihydroceramide caused less than a 10% decline in cell
number. As shown in Fig. 5, cell death
induced by addition of C6-ceramide to sup (
) cells in low
serum was apoptotic. Addition of low serum alone was unable to induce
apoptosis of sup (
) cells (Fig. 5, lane 1); however, supplementing the low serum treatment with 10-50 µM ceramide was able to induce apoptosis, as indicated by lanes 2-4.
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C6-ceramide induces cell cycle arrest in 10% serum.
Cell cycle analysis showed that ceramide treatment in the presence of
10% serum led to a pronounced arrest of cells in the G2/M
phase of the cell cycle (Table 1). While
control cultures had ~17-19% of the population in
G2/M, C6-ceramide-treated cells showed as many
as 28-33% of cells in G2/M. The G2/M
arrest is apparent in both the sup () and sup (+) cell lineages and
is specific to C6-ceramide. Thus the closely related lipid
dihydroceramide, which differs from ceramide only by the lack of a
double bond between carbons-4 and -5 of the sphingoid backbone, had
little effect on cell number or on cell cycle.
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DISCUSSION |
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The current study begins to define a relationship between calcium and ceramide in apoptotic signaling. Several groups have reported that an early, sustained increase in cytoplasmic free calcium ([Ca2+]i) is a necessary precedent to cell death (20, 34). Others have reported that a decrease (2, 26, 31) or no detectable change in cytoplasmic calcium (30) correlates with apoptosis. We have shown previously that low serum-induced apoptosis in sup (+) cells is not accompanied by a measurable increase in [Ca2+]i (26). Because of the long time course needed for initiation of apoptosis, it is difficult to rule out a transient or localized change in [Ca2+]i that could be missed. [Ca2+]i is only measured at discrete times because the calcium indicator used to measure [Ca2+]i (fura 2) leaks out of these cells in ~1 h. In contrast to the inconsistent reports regarding the role of cytoplasmic calcium in apoptosis, a number of studies suggest that changes in ER calcium are important in the regulation of apoptosis (2, 16, 26). We have shown previously (12, 26) that a decrease in ER calcium occurs before apoptosis of sup (+) cells. Furthermore, we found that if we block the decrease in ER calcium by raising extracellular calcium, we block apoptosis. However, because released ER calcium must, at least transiently, enter the cytosol, it is difficult to distinguish whether it is the decrease in ER calcium per se or the transient or localized increase in cytoplasmic calcium that is important for signaling apoptosis. However, because of the reproducibility of the decrease in ER calcium and because we can block the decrease in ER calcium, and consequently apoptosis, by raising extracellular calcium, we use altered ER calcium as a marker of altered calcium homeostasis.
Although a number of studies have shown that sphingosine and other
mitogenic lysosphingolipids can modulate calcium homeostasis, only a
few studies have shown an interconnection between ceramide and
intracellular calcium. These studies postulate a role for ceramide
upstream of calcium changes (17, 21). Lepple-Wienhues et
al. (17) have reported that addition of
C2-ceramide or C6-ceramide to T lymphocytes
blocks store-operated calcium entry currents measured by patch clamp.
Based on these studies, we initially hypothesized that an increase in
ceramide might be responsible for the altered calcium homeostasis that
occurs during apoptosis of sup (+) cells. We have previously shown that
the sustained decrease in ER calcium observed before apoptosis was
secondary to a decrease in store-operated calcium entry
(26). We therefore hypothesized that an increase in
ceramide might precede alterations in calcium homeostasis after low
serum stimulation of sup (+) cells. However, we found instead that an
increase in ceramide occurs well after the observed alterations in
calcium. In fact, the current study is unique in suggesting a converse
interaction between calcium and ceramide. Based on temporal
relationships and pharmacology, we present data showing that changes in
calcium homeostasis occur before changes in ceramide during apoptosis of sup (+) cells. Changes in calcium homeostasis, as evidenced by
decreased ER calcium, occur after 4 h of low serum treatment. In
contrast, an elevation in ceramide is evident only after 16 h.
Temporally therefore, a decrease in ER calcium precedes ceramide elevation during low serum-induced apoptosis of sup (+) cells. Furthermore, pharmacological manipulation of ER calcium levels leads to
a parallel modulation of ceramide levels. When ER calcium levels are
depleted with thapsigargin treatment, ceramide elevation is observed in
sup (+) cells. Conversely, when ER calcium levels are prevented from
decreasing [e.g., with high calcium treatment in conjunction with low
serum in sup (+) cells], ceramide levels remain at basal levels.
Blocking the decrease in ER calcium blocks both the rise in ceramide
and apoptosis. However, raising extracellular calcium in sup (+) cells
treated with low serum does not block cell death; rather, it shifts
cell death from apoptotic to necrotic in conjunction with blocking the
rise in ceramide. We also find that sup () cells in low serum, a
condition that does not result in apoptosis, can be made to undergo
apoptosis by the addition of C6-ceramide. It has been
suggested that C6-ceramide might act more like a
lysophospholipid and have effects not related to the specific effects
of ceramide (8). Although one must always be cautious in
using pharmacological analogs, numerous studies have shown the validity
of using C6-ceramide as an analog of ceramide (see Ref.
24). Furthermore, in this study we show that inactive C6-dihydroceramide did not mimic the effects of
C6-ceramide, thereby reducing the probability of
nonspecific effects.
The data in this manuscript are consistent with data suggesting that changes in ceramide occur late in apoptosis (33, 35, 36, 41). There are several reports suggesting that activation of initiator caspases is involved in the generation of ceramide. Tepper et al. (36) report that generation of ceramide is blocked by zVAD-fmk, an inhibitor of caspase 9, an initiator caspase, but ceramide generation is not blocked by DEVD-CHO, an inhibitor of the effector caspases. These authors suggest that generation of ceramide is associated with the activation of effector caspases or the propagation of apoptosis. Interestingly, Nakagawa et al. (22) recently reported that ER stress, such as that induced by treatment with thapsigargin, causes activation of caspase 12, a caspase that is also inhibited by zVAD-fmk.
Having ordered the pathway to show that the changes in calcium precede changes in ceramide raises the question of how altered ER calcium might influence ceramide levels. It is intriguing that the enzymes involved in de novo synthesis of ceramide are located at the ER membrane (7, 18). Thus it is possible that the level of regulation derives from the availability of calcium to the biosynthetic enzymes responsible for ceramide generation.
Another complexity of the ceramide signal arises from the differential
effects that occur, depending on the context of the signal. Unlike the
decrease in ER calcium, addition of C6-ceramide alone does
not result in apoptosis in sup (+) or sup () cells in 10% serum.
Only when additional stress is present (e.g., in low serum conditions)
does C6-ceramide treatment lead to apoptosis. In the
absence of an additional stress signal, C6-ceramide
treatment results in cell cycle arrest. This result is similar to
another model system of serum deprivation-induced apoptosis, the MOLT-4 cell line, in which the ability of ceramide to induce apoptosis rather
than cell cycle arrest depends on the level of DAG present in the cell
(11). When DAG levels were high, ceramide treatment led to
cell cycle arrest (in the case of the MOLT-4 cells,
G0/G1 arrest). However, when DAG levels were
not elevated, ceramide treatment resulted in apoptosis. It has been
suggested (29) that ceramide induces apoptosis by
stimulation of a phosphatase that dephosphorylates Bcl-2 and that DAG
opposes the action of ceramide by stimulation of protein kinase
C-
-dependent phosphorylation of Bcl-2. Thus a high DAG would be
expected to oppose ceramide-induced apoptosis. However, we found that
the DAG level in 10% serum, conditions where exogenous ceramide
treatment led to cell cycle arrest, was not significantly different
from that measured in low serum (conditions where ceramide treatment
led to apoptosis). Thus, unlike the MOLT-4 system in which the ratio of
ceramide to DAG determines apoptosis vs. cell cycle arrest, in the
hamster model the additional signal modulating the effect of ceramide remains to be determined. It is possible that the additional stress signal could modify the pathways that metabolize ceramide and thereby
alter its effect, as has been suggested in other systems (35).
The data in this study suggest that changes in ceramide occur after
alterations in calcium homeostasis and further show that inhibition of
altered calcium homeostasis blocks the rise in ceramide and apoptosis.
A role for ceramide in apoptosis is supported by the observation that
addition of ceramide to sup () cells in low serum can trigger
apoptosis. The data show that an elevation in ceramide is a late event
and is downstream of altered calcium homeostasis in apoptotic signaling.
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ACKNOWLEDGEMENTS |
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We thank John G. Petranka for technical assistance and Drs. Cynthia A. Afshari and Darryl C. Zeldin for careful review of the manuscript.
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FOOTNOTES |
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Present address for S. Jayadev: Toxicology Dept., Boehringer Ingelheim Pharmaceuticals Inc., PO Box 368, Ridgefield, CT.
Address for reprint requests and other correspondence: E. Murphy, NIEHS, P. O. Box 12233, MD D2-03, RTP, NC 27709 (E-mail: murphy1{at}niehs.nih.gov).
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.
Received 22 January 2000; accepted in final form 8 June 2000.
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