 |
INTRODUCTION |
The sphingomyelin (SM)1
pathway is an ubiquitous signaling system conserved from yeast to
humans (1-3). Ceramide is the central molecule in this pathway,
generated from SM by the action of a neutral sphingomyelinase or acid
sphingomyelinase (ASMase) or by de novo synthesis
coordinated through the enzyme ceramide synthase. Ceramide has been
implicated as second messenger for cytokines signaling through the
tumor necrosis factor (TNF) receptor superfamily and for diverse
stresses (e.g. ionizing radiation, oxidative stress, ultraviolet C, and heat). Although in some cells ceramide may provide
proliferative or differentiating signals, often the outcome of ceramide
signaling is the induction of apoptosis. Evidence that ceramide is
involved directly in the induction of apoptosis is summarized as
follows: (i) agonist-induced ceramide elevations precede biochemical
and morphological manifestations of apoptosis (4-7); (ii) elevation of
cellular ceramide levels using natural ceramide (8-10), ceramide
analogs, or exogenous SMases mimic the effects of stress on apoptosis
(11-16); (iii) pharmacological agents which interfere with enzymes of
ceramide metabolism and elevate cellular ceramide, such as the
glucosylceramide synthase inhibitor D-threo-1-phenyl-decanoylamino-3-morpholino-1-propanol or
the ceramidase inhibitor N-oleoylethanolamine, uniformly
enhance apoptosis (17-19), whereas agents that reduce ceramide
generation, such as the ceramide synthase inhibitor fumonisin B1,
prevent apoptosis (10, 20, 21); and (iv) other lipids, including
other sphingolipid metabolites (except perhaps sphingosine), fail to
consistently signal apoptosis (12, 13, 16).
The most compelling evidence supporting ceramide as mediator of
apoptosis is derived, however, from data using genetic models. Lymphoblasts from Niemann-Pick disease (NPD) patients, which have an
inherited deficiency of ASMase activity, failed to respond to ionizing
radiation with ceramide generation or apoptosis (22-24). These
abnormalities were, however, reversed by transduction of the human
asmase cDNA. Furthermore, asmase knockout
mice also manifested defects in radiation-induced ceramide generation
and apoptosis in endothelium of the lung and throughout the central nervous system (22, 25). asmase knockout mice were, however, normally sensitive to radiation-induced apoptosis in thymic cells. In
contrast, p53 knockout mice were largely insensitive in the thymus but
were not resistant to radiation-induced apoptosis in endothelium. Since
these original findings, other reports have confirmed that the
Niemann-Pick cells are resistant to ionizing radiation (23, 24).
Whether the ASMase-deficient systems are resistant to
Fas-/APO-1-/CD95-induced death has been a matter of debate. Testi and
coworkers (26) claimed the Niemann-Pick cells manifested
resistance to Fas-induced death, whereas Borst and coworkers (27) found
no differences. Recently, Green and coworkers (28) reported partial
resistance to Jo2 anti-Fas-induced liver failure and death in
asmase knockout mice in vivo.
These genetic models have not uniformly been accepted as definitive.
The NPD lymphoblasts described above were transformed with Epstein-Barr
virus, and the transformation process itself, as well as known
Epstein-Barr virus strain-specific phenotypic differences
(29-31), might have played an unknown role in the radiation resistance. Furthermore, the potential for a direct contribution of the
mutated ASMase protein to the radiation resistance phenotype has not
been assessed. Additionally, it remains possible that the resistance of
endothelium to radiation-induced apoptotic death in vivo is
not a cell autonomous defect but rather reflects a systemic process. To
address these issues directly, we have generated murine embryonic
fibroblasts (MEFs) from the asmase knockout mice. We observe
complete resistance to radiation-induced apoptosis in these cells,
partial resistance to serum withdrawal or TNF
/ actinomycin D (ActD)
treatment, yet complete sensitivity to staurosporine-induced apoptosis.
Furthermore, sensitivity to radiation-, serum withdrawal-, and
TNF
/ActD-induced death could be restored by pretreatment with minute
amounts of natural ceramide. Rescuing the apoptotic phenotype without
reversing the genotype by the product of the enzymatic deficiency
indicates that ceramide is obligate, in particular for radiation, for
apoptotic death.
 |
MATERIALS AND METHODS |
Reagents--
Actinomycin D, Hoechst-33258, and staurosporine
were from Sigma. Recombinant mouse TNF
was from Pharmingen. The
fluorogenic substrate Ac-DEVD-AFC was purchased from Kamiya
Biomedical Co. Anti-phospho-mitogen-activated protein kinase and
anti-phospho-p38 antibodies were from New England Biolabs, whereas the
anti-phospho c-Jun N-terminal kinase (JNK) antibody was from
Promega. The anti-TNF receptor (TNFR) antibody was purchased from Santa
Cruz Biochemicals. LK6D Silica Gel 60A TLC plates were from Whatman.
C16-ceramide and C16-dihydroceramide were from Biomol.
MEF Preparation and Culture--
MEFs derived from
asmase+/+ and
asmase
/
day 12-13 embryos were
prepared as described (32). Cells were subcultured every 3 days for a
maximum of six passages. Only MEFs from passages 2-5 were used for
experiments. 0.05-0.1 × 106 cells were seeded per
60-mm plate and grown in Dulbecco's modified Eagle's medium
supplemented with 15% fetal bovine serum (FBS) at 37 °C in a 5%
CO2 atmosphere. Unless otherwise indicated, cells were made
quiescent by 12-h incubation in 0.5% FBS and then either irradiated
using a 137Cs Shepherd Mark-I irradiator or treated with
different inducers of apoptosis, as stated in the figure legends. For
ceramide rescue, cells were pretreated for 30 min with
C16-ceramide or C16-dihydroceramide in dodecane:ethanol (2:98, v/v; 0.05% final concentration).
Lipid Mass Analysis--
Total cellular lipids were extracted as
described previously (13). To measure SM mass, equal aliquots of each
sample were spotted on TLC plates, and SM was resolved using
chloroform:methanol:acetic acid:water (50:30:8:3, v/v/v/v) as solvent.
After chromatography, plates were sprayed with 35% (v/v) sulfuric
acid, and lipids were charred by heating in an oven at 180 °C for 30 min and quantified by comparison with a concomitantly run standard
curve composed on known quantities of SM. Ceramide was quantified by
the diacylglycerol kinase assay as described previously (33). ASMase
and neutral sphingomyelinase activities were measured as described by
Lin et al. (34).
Bisbenzimide Staining--
At different times after treatment,
cells were processed for nuclear staining as described (35). Briefly,
the medium containing the floating dead cells was aspirated and pooled
together with a PBS wash of the monolayer in a 15-ml Falcon conical
tube. The remaining adherent cells were trypsinized, collected in the
same tube, and centrifuged at 400 × g for 5 min. The
pellets were gently vortexed and fixed in 400 µl of 10% buffered
formalin phosphate for 10 min at room temperature. Nuclear apoptosis
was assessed by staining of formalin-fixed cells with the DNA binding
fluorochrome Hoechst-33258 as described (13). A minimum of 200 cells
were scored at each point.
Caspase 3 Assay--
The fluorogenic substrate Ac-DEVD-AFC was
used to measure caspase 3 activity according to the manufacturer's
instructions. Briefly, cells were lysed in radioimmunoprecipitation
assay buffer (50 mM Tris-HCl, pH 7.6, 150 mM
NaCl, 1% Triton X-100, 0.1% SDS, and 0.5% sodium deoxycholate plus
10 µg/ml leupeptin and 10 µg/ml soybean trypsin inhibitor), and the
protein concentration was measured. 20 µg of cell lysate were mixed
with 8 µM Ac-DEVD-AFC in 200 µl of caspase 3 buffer (50 mM HEPES, 10% sucrose, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and 10 mM dithiothreitol) in a 96-well plate. Fluorescence
generated by the release of the fluorogenic group AFC on
cleavage by caspase 3 was measured in a PerkinElmer LS50B luminescence
spectrometer by excitation at 400 nm and emission at 505 nm.
Immunoblot Assays--
Mitogen-activated protein kinase, JNK,
and p38 activation were detected by Western blot with
anti-phospho-specific antibodies. After 12 h in 0.5% FBS, cells
were stimulated with 100 ng/ml epidermal growth factor, 100 ng/ml
platelet-derived growth factor, or 100 ng/ml TNF
for 10 min, washed
with PBS, and lysed in 0.2 ml of Nonidet P-40 lysis buffer (20 mM Tris-HCl, 137 mM NaCl, 2 mM
EDTA, 10% glycerol, and 1% Nonidet P-40 plus protease and phosphatase inhibitors). Western blots were performed as described (36).
 |
RESULTS |
Characterization of asmase
/
MEFs--
To
investigate whether ASMase-deficient cells displayed cell autonomous
defects in apoptosis, we generated MEFs from wild-type and
ASMase-deficient mice. No measurable ASMase activity was detected in
the asmase
/
MEFs (Table
I), whereas neutral sphingomyelinase
activity was unchanged. As a consequence of the ASMase deficiency,
asmase
/
MEFs manifested a modest
20% increase in the SM content and a 12% decrease in ceramide levels
(Table I). Both wild-type and knockout MEFs grew at similar rates
(Table I) and manifested identical morphology (data not shown). The
content and distribution of caveolin-1, the primary protein within the
sphingolipid-rich microdomains termed caveolae, were similar in
asmase+/+ and
asmase
/
MEFs (37). Furthermore,
the proportion of detergent-insoluble caveolar membranes isolated and
the signaling components found within these membranes appeared similar
in asmase+/+ and
asmase
/
MEFs (37). Activation of
p38, c-Jun N-terminal, and extracellular signal-regulated
kinases in response to platelet-derived growth factor, TNF
, and
epidermal growth factor were also identical in
asmase+/+ and
asmase
/
MEFs (see Table I and
Fig. 3B). Thus, by numerous criteria, asmase+/+ and
asmase
/
MEFs appeared
similar.
asmase
/
MEFs Are Resistant to Radiation-induced
Apoptosis--
Our previous studies demonstrated that NPD
lymphoblasts failed to respond to ionizing radiation with ceramide
generation or apoptosis (22-24). To evaluate whether
asmase
/
MEFs display similar
defects, cells were made quiescent by incubation in 0.5% FBS for
12 h, irradiated, and assessed for apoptosis at various times
after irradiation. A 12-h quiescence period was selected to minimize
the basal level of apoptosis induced by serum withdrawal, which under
these conditions was typically in the range of 4-5%. Fig.
1A, left panel,
shows that exposure of asmase+/+ cells to a dose
of 20 Gy resulted in time-dependent apoptosis, as measured
by bisbenzimide staining. Apoptosis was apparent by 3 h and
maximal 12 h after irradiation, when 25-30% of the population manifested chromatin condensation or nuclear segmentation, or both,
morphologic characteristics of apoptotic cells. In contrast, asmase
/
cells were resistant to
radiation-induced apoptosis (Fig. 1A) for as long as 72 h (data not shown). To determine whether a higher radiation dose might
induce apoptosis in the asmase
/
cells, we treated the wild-type and knockout cells with increasing radiation doses and measured apoptosis after 24 h (Fig. 1A,
right panel). As little as 1 Gy increased apoptosis to 13% of the
asmase+/+ population, and a maximal effect was
obtained with a 20-Gy dose. Quantitatively similar results were
reported by Santana et al. (22) after irradiation of
Epstein-Barr virus-transformed lymphoblasts from Niemann-Pick patients.
In contrast, asmase
/
MEFs were
resistant to radiation up to 30 Gy.

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Fig. 1.
asmase / MEFs
are resistant to radiation-induced apoptosis. A,
asmase+/+ and asmase /
MEFs were grown in 0.5% FBS for 12 h, irradiated, and apoptosis
was measured by bisbenzimide staining at various times after a 20 Gy
irradiation (left) and at 24 h after irradiation with
various doses (right). Data (mean ± S.E.) represent
three studies performed in duplicate. At least 200 cells were evaluated
for each point. B, time course of caspase 3-like activity
after 20 Gy measured as cleavage of the fluorogenic substrate
Ac-DEVD-AFC. Cells were made quiescent, irradiated as in A,
and then cell lysates were prepared in radioimmunoprecipitation assay
buffer, and the DEVDase activity was measured. Data (mean ± S.E.) represent three studies performed in duplicate. C,
ceramide levels in asmase+/+ and
asmase / MEFs at 15 min after 20 Gy of irradiation were determined by the diacylglycerol kinase assay.
Data (mean ± S.E.) represent three independent studies performed
in duplicate. *, p < 0.05 versus
unirradiated controls or irradiated
asmase / MEFs.
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|
To confirm the results of Fig. 1A using a biochemical assay,
caspase 3 activity was measured in both
asmase+/+ and
asmase
/
MEFs at various times
after irradiation. As shown in Fig. 1B, we detected a
2.8 ± 0.1-fold increase in caspase activity at 6 h after 20 Gy irradiation (p < 0.001 versus zero time
control) in asmase+/+ MEFs and a maximal
3.4 ± 0.1-fold increase at 10 h. In agreement with the
results shown in Fig. 1A, we could not detect any increase in caspase 3 activity in the
asmase
/
MEFs at any time point.
These results show that lack of ASMase renders cells resistant to
radiation-induced apoptosis.
To confirm that ASMase deficiency resulted in resistance to ceramide
generation, ceramide levels were measured after irradiation of
asmase+/+ and
asmase
/
MEFs. In
asmase+/+ cells, the ceramide content increased
1.5-fold by 15 min after 20 Gy irradiation (Fig. 1C;
p < 0.05) and returned to baseline within 1 h. In
contrast, no ceramide elevation was observed in the
asmase
/
MEFs under the same
conditions (Fig. 1C) at any time point up to 4 h. These
data indicate that, as in NPD cells, lack of ASMase renders MEFs
resistant to radiation-induced ceramide generation and apoptosis.
Although these studies are consistent with a role for ceramide
accumulation in the apoptotic response to ionizing radiation, they do
not constitute proof that the apoptosis resistance resulted from lack
of ceramide production.
asmase
/
MEFs Are Sensitive to Staurosporine-induced
Apoptosis--
To evaluate whether the mutation leading to the
asmase
/
phenotype resulted in a universal
inactivation of the apoptotic apparatus, MEFs were exposed to another
type of proapoptotic stress. Staurosporine is a potent protein kinase
inhibitor (38) that induces apoptosis in many cell types (39-41). In
the wild-type MEFs, a dose as low as 0.01 µM
staurosporine induced apoptosis in 50% of the population after 24 h, whereas 95% apoptosis was observed with a dose of 0.1 µM staurosporine (Fig.
2A, left panel). In contrast
to the irradiation experiments, the
asmase
/
MEFs showed no
resistance to staurosporine-induced cell death, and the level of
apoptosis was essentially identical to that of the
asmase+/+ MEFs. In fact, if there was any
difference, the asmase
/
MEFs
died slightly faster (Fig. 2A, right panel); at 6 h
after addition of staurosporine 37.6 ± 1.7% of the
asmase
/
MEFs were undergoing
apoptosis, whereas only 24.6 ± 0.1% of the asmase+/+ cells displayed apoptosis. To confirm
these results, caspase 3 activation was also measured in the
staurosporine-treated MEFs. By 8 h after treatment, we detected an
~6.5-fold increase in caspase 3 activity in the
asmase+/+ MEFs, in good agreement with
staurosporine as a more potent inducer of apoptosis than ionizing
radiation in MEFs (compare Fig. 1A with Fig. 2A).
Caspase 3 activation in asmase
/
MEFs was very similar to that in the asmase+/+
MEFs (Fig. 2B). In contrast to radiation,
staurosporine-induced apoptosis did not appear associated with ceramide
generation. Staurosporine failed to generate ceramide at any time
within the first 30 min of treatment in either
asmase+/+ or
asmase
/
MEFs (data not shown).
These studies indicate that
asmase
/
MEFs are fully competent
in generating an apoptotic response and suggest that the lack of
apoptosis to radiation may be specifically associated with
ASMase deficiency.

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Fig. 2.
Staurosporine induces apoptosis in
asmase / MEFs. A,
staurosporine dose response at 24 h (left) and time
course using 0.04 µM staurosporine (right) of
apoptosis measured by bisbenzimide staining as in Fig. 1A.
Data (mean ± S.D.) are from two experiments performed in
duplicate. B, increase in caspase activity after 0.04 µM staurosporine treatment as measured in Fig.
1B. Data (mean ± S.D.) are from one representative of
three independent studies performed in duplicate.
|
|
asmase
/
MEFs Are Partially Resistant to Apoptosis
after TNF
/ActD Treatment--
Whereas ASMase activation and
ceramide accumulation have been reported in response to TNF
in
numerous cell types (12, 35, 42), studies were carried out to explore
whether asmase
/
MEFs showed a
defect in TNF
responsiveness. Many cell types, including MEFs,
require previous sensitization for TNF
to become toxic (43-45). As
in previous studies, a maximal dose of TNF
(1 ng/ml) was unable to
signal apoptosis in wild-type MEFs. However, the combination of TNF
and ActD effectively induced time- and dose-dependent
apoptosis. As little as 0.2 µg/ml ActD in combination with 1 ng/ml
TNF
induced apoptosis in 50% of the
asmase+/+ MEFs (Fig.
3A) after 24 h, while 75%
apoptosis was achieved with 0.3% µg/ml ActD and TNF
. Apoptosis of
asmase+/+ MEFs in response to TNF
/ActD was
detectable by 8 h and maximal at 24 h of treatment (data not
shown). At all times from 8 to 24 h the apoptotic response of
asmase
/
MEFs to TNF
/ActD was
reduced by 25-50% compared with asmase+/+ MEFs
(shown in Fig. 3A at 24 h), indicating partial
resistance to this stress.
asmase
/
MEFs were also resistant
to ceramide generation. In contrast to the transient ceramide elevation
in asmase+/+ MEFs after ionizing radiation (Fig.
1C), TNF
(1 ng/ml)/ActD (0.3 µg/ml) induced a
persistent 40-50% ceramide increase beginning 15 min after
irradiation and lasting for at least 1 h.
asmase
/
MEFs, however, displayed
no ceramide elevation up to 6 h after irradiation. These data
suggest that only part of the apoptotic response to TNF
/ActD is
mediated via ASMase.

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Fig. 3.
TNF -dependent apoptosis
in asmase /
MEFs. A, MEFs were treated with TNF
(1 ng/ml) and increasing concentrations of ActD, and apoptosis was
measured after 24 h as in Fig. 1A. Data (mean ± SD) are from one representative of three independent studies performed
in duplicate. B, 30 µg of total cell lysates prepared from
asmase+/+ and
asmase / MEFs were resolved by
7.5% SDS-polyacrylamide gel electrophoresis. After transfer to a
nitrocellulose membrane, TNFR expression was analyzed by Western blot
with an anti-TNFR antibody (left panel). To measure JNK,
p38, and mitogen-activated protein kinase activation, cells were grown
in low serum (0.5% FBS) for 24 h and then either left untreated
or stimulated with 100 ng/ml TNF , epidermal growth factor, or
platelet-derived growth factor for various times. Cell lysates were
prepared, and 50 µg of protein from each sample were analyzed by
Western blot with anti-phospho-specific antibodies (right
panel). Note that the anti-phospho-JNK antibody recognizes both
the JNK1 and JNK2 activated isoforms. In the phospho-extracellular
signal-regulated kinase studies, cells were stimulated for 10 min.
Similar results were obtained in two independent experiments.
|
|
To exclude the possibility that the difference in the response of
asmase+/+ and
asmase
/
MEFs to TNF
/ActD was
attributable to a deficiency in TNF receptors, we examined the level of
TNFRs by Western blot with an anti-TNFR antibody. Fig. 3B, left
panel, shows that asmase+/+ and
asmase
/
MEFs express similar
levels of TNFRs. Despite similar receptor levels, it was possible that
TNFRs might not be functional in asmase
/
MEFs. To rule out this
possibility, we treated asmase+/+ and
asmase
/
MEFs with TNF
and
examined the status of three major signaling pathways, the JNK, p38,
and extracellular signal-regulated kinase 1 and 2 cascades, with
antibodies specific for the activated kinases. Fig. 3B, right
panel, shows that TNF
induced a robust activation of C-terminal
Jun and p38 protein kinases in both types of MEFs after 10 min of
treatment. We did not detect extracellular signal-regulated kinase 1 and 2 activation by TNF
, although it was rapidly activated by growth
factors such as epidermal growth factor and platelet-derived growth
factor in both asmase+/+ and
asmase
/
MEFs (Fig.
3B). Nuclear factor
B nuclear translocation in response to TNF
treatment was also normal in ASMase-deficient MEFs (Table I).
These results suggest that the differences in apoptosis observed between asmase+/+ and
asmase
/
MEFs are not
attributable to an alteration in the level or the functionality of
membrane receptors but rather to a selective deficiency in ASMase activity.
asmase
/
MEFs Are Partially Resistant to Serum
Withdrawal-induced Apoptosis--
To investigate the response of
ASMase-deficient MEFs to induction of apoptosis by serum
withdrawal, MEFs from wild-type and knockout animals were plated in
15% FBS for 24 h and then switched to low serum. After 48 h,
wild-type MEFs displayed more apoptosis than
asmase
/
at all concentrations
from 0.1 to 4% FBS (Fig. 4). Differences were more pronounced in cells challenged with very low doses of serum
(<1%). (Note that in all previous experiments, cells were serum
deprived with 0.5% FBS for only 12 h before any treatment.)

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Fig. 4.
Low serum-induced apoptosis in
asmase / MEFs.
Cells were grown for 24 h in 15% FBS and then switched to low
serum (0.1-4% FBS). After 48 h, cells were analyzed for
apoptosis as in Fig. 1A. Data (mean ± S.D.) are from
two independent studies performed in duplicate.
|
|
Natural Ceramide Can Revert the Apoptotic Response to Radiation in
the asmase
/
MEFs--
To provide definitive evidence
that the lack of ceramide is the cause of the defects in apoptosis, we
pretreated asmase
/
cells with natural
C16-ceramide before apoptosis induction. Fig. 5 shows that low doses of
C16-ceramide (0.05-1.0 µM) had no direct effect on apoptosis at 24 h of treatment. In fact, doses up to 1 µM C16-ceramide were without effect for as
long as 48 h (data not shown). However, these doses completely
restored apoptosis in response to 20 Gy of ionizing radiation
(XRT + 0.5 or 1.0 µM C16-ceramide,
p < 0.001 versus 0.5 or 1.0 µM C16-ceramide alone). In contrast, up to
1.0 µM C16-dihydroceramide, the precursor of C16-ceramide, was without effect (data not shown),
indicating a specificity for ceramide in this response. Similarly,
C16-ceramide rescued the apoptosis defects of
asmase
/
MEFs after treatment
with TNF
/ActD and serum withdrawal (data not shown).

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Fig. 5.
Natural C16-ceramide restores
radiation sensitivity to
asmase / MEFs. For
these studies, cells were pretreated for 30 min with
C16-ceramide or diluent (0.05% final concentration) before
irradiation with 20 Gy (XRT). Apoptosis was evaluated in 300 cells per
point as in Fig. 1A after 24 h. 95% confidence limits
for each point are shown in one representative of three independent
experiments.
|
|
 |
DISCUSSION |
The present investigations provide definitive evidence for cell
autonomous defects in apoptosis in ASMase-deficient cells. Similar to
the defects observed in endothelial apoptosis in the lung (22) and
throughout the central nervous system (25), MEFs from
asmase
/
animals failed to
generate ceramide within the first 30 min of ionizing radiation
treatment and displayed almost complete resistance to induction of
apoptosis. In contrast to the marked resistance to ionizing radiation,
the sensitivity to staurosporine, which appears to be ceramide
independent, was unchanged in
asmase
/
MEFs. Thus, resistance
to apoptosis in asmase
/
MEFs was
not global but rather stress type specific. Consistent with this
notion, asmase
/
MEFs displayed partial
resistance to TNF
/ActD and serum withdrawal. The fact that apoptosis
resistance of asmase
/
MEFs to
radiation, and the partial resistance to TNF
/ ActD, could be
overcome by natural ceramide provides compelling evidence that it is
the lack of ceramide and not the lack of ASMase that determines apoptosis sensitivity. The ability to rescue the apoptotic phenotype without reversing the genotype by providing the product of the enzymatic deficiency is proof that ceramide is obligate for apoptosis induction in cells that use the SM pathway.
The present investigations extend our knowledge of the mechanisms of
radiation-induced apoptosis. Our studies originally proposed that in
addition to double-stranded breaks, damage to the plasma membrane and
the generation of the proapoptotic lipid ceramide signaled apoptosis in
some cell types (13). This was supported by the demonstration that
ionizing radiation was capable of SMase activation in isolated
membranes devoid of DNA altogether. Since these initial studies,
numerous reports confirm the early generation of ceramide in response
to irradiation (14, 23, 46-55) and by a variety of biochemical,
pharmacological, and genetic approaches have provided support to the
critical role of this event in the ensuing apoptotic response (14, 22,
23, 35, 46, 48, 54, 55). Here, we show that ionizing radiation-induced
apoptosis of MEFs requires at least two components, one of which is
ceramide, as doses of natural ceramide that alone are without effect
restore apoptosis to irradiated cells. Recent studies by Zundel
and Giaccia (23) and Zundel et al. (24) may shed light on
the mechanism of this ceramide-dependent step. These
investigators reported that ionizing radiation induced
ceramide-dependent complexation of the antiapoptotic
protein phosphoinositide 3-kinase to caveolin-1, resulting in
phosphoinositide 3-kinase inactivation. Whether the ceramide-dependent step in MEFs requires phosphoinositide
3-kinase inactivation and the nature of the ceramide-independent step
require further investigation.
Nix and Stoffel (56) recently reported marked biochemical alterations
and membrane dysfunction in cells derived from their asmase
knockout mice, which were generated separately from our colony.
Although both knockouts are maintained in a C57BL/6 × 129 background, the substrains of the 129 line are different (22, 57, 58).
Furthermore, these ASMase-deficient mouse lines were developed using
nonisogenic stem cell clones (22, 57, 58). MEFs derived from their mice
displayed dramatic reduction in caveolin content and resistance to
isolation of caveolae (56). Further, hepatocytes displayed a 2-fold
increase in plasma membrane SM content accompanied by reduced signaling
through tyrosine kinases in T lymphocytes, lymphopenia, the absence of
proliferation in response to anti-CD3, reduced expression of the
antiapoptotic adaptor FLIP, and a paradoxic increase in
apoptosis of anti-CD3 pretreated splenocytes upon activation of CD95
(56). The conclusion of these studies was that disruption of membrane
microdomains in response to altered sphingolipid metabolism has
significant impact on signaling. We have repeated all of these studies
and do not find any of these abnormalities in cells derived from our asmase knockout colony (Ref. 37 and Table I). Phenotypic
differences also exist between the two asmase knockout mouse
colonies. While their mice die from NPD by 4 months of age (57), our
animals do not manifest even the earliest sign of NPD, a resting
tremor, until 12-16 weeks of age and survive to 9.6 ± 0.4 (mean ± S.D.) months of age (22, 25, 37). Extragenic
strain-specific enhancers and suppressors, such as polymorphisms or
background mutations, and epigenic environmental factors are well known
to modify the expression of genetic phenotypes in mice. Whatever the
reason for the differences, it is clear that the asmase
knockout mice of Stoffel and coworkers (56, 57) develop NPD more
rapidly than our knockout mice. For these reasons, we urge that
investigators interested in using these models to study apoptosis
select reagents carefully.
The present investigations demonstrate cell autonomous defects in
apoptosis in ASMase-deficient cells. These defects are stress type
specific; although MEFs were completely resistant to ionizing radiation, they were only partially resistant to TNF
/Act D or serum
withdrawal, and not at all resistant to staurosporine. Furthermore, the
ability of natural ceramide to rescue the apoptosis phenotype without
reversing the genotype provides evidence that ceramide is obligate for
induction of apoptosis in some stress response systems.